Mammalian cytokines; related reagents

ABSTRACT

Purified genes encoding a cytokine or composite cytokine from a mammal, reagents related thereto including purified proteins, specific antibodies, and nucleic acids encoding these molecules are provided. Methods of using said reagents and diagnostic kits are also provided.

This filing is a divisional of U.S. patent application Ser. No.10/000,776, filed Nov. 30, 2001, now issued as U.S. Pat. No. 7,148,300,which is a continuation-in-part of U.S. patent application Ser. No.09/791,497, filed Feb. 22, 2001, now abandoned which is acontinuation-in-part of U.S. patent application Ser. No. 09/627,897,filed Jul. 27, 2000, now abandoned and claims benefit from U.S.Provisional Patent Application Nos. 60/146,581, filed Jul. 30, 1999, and60/147,763, filed Aug. 6, 1999, each of which are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The present invention pertains to compositions related to proteins whichfunction in controlling biology and physiology of mammalian cells, e.g.,cells of a mammalian immune system. In particular, it provides purifiedgenes, proteins, antibodies, and related reagents useful, e.g., toregulate activation, development, differentiation, and function ofvarious cell types, including hematopoietic cells.

BACKGROUND OF THE INVENTION

Recombinant DNA technology refers generally to the technique ofintegrating genetic information from a donor source into vectors forsubsequent processing, such as through introduction into a host, wherebythe transferred genetic information is copied and/or expressed in thenew environment. Commonly, the genetic information exists in the form ofcomplementary DNA (cDNA) derived from messenger RNA (mRNA) coding for adesired protein product. The carrier is frequently a plasmid having thecapacity to incorporate cDNA for later replication in a host and, insome cases, actually to control expression of the cDNA and therebydirect synthesis of the encoded product in the host.

For some time, it has been known that the mammalian immune response isbased on a series of complex cellular interactions, called the “immunenetwork”. Recent research has provided new insights into the innerworkings of this network. While it remains clear that much of theresponse does, in fact, revolve around the network-like interactions oflymphocytes, macrophages, granulocytes, and other cells, immunologistsnow generally hold the opinion that soluble proteins, known aslymphokines, cytokines, or monokines, play a critical role incontrolling these cellular interactions. Thus, there is considerableinterest in the isolation, characterization, and mechanisms of action ofcell modulatory factors, an understanding of which will lead tosignificant advancements in the diagnosis and therapy of numerousmedical abnormalities, e.g., immune system disorders. Some of thesefactors are hematopoietic growth and/or differentiation factors, e.g.,stem cell factor (SCF) or IL-12. See, e.g., Mire-Sluis and Thorpe (1998)Cytokines Academic Press, San Diego; Thomson (ed. 1998) The CytokineHandbook (3d ed.) Academic Press, San Diego; Metcalf and Nicola (1995)The Hematopoietic Colony Stimulating Factors Cambridge University Press;and Aggarwal and Gutterman (1991) Human Cytokines Blackwell.

Lymphokines apparently mediate cellular activities in a variety of ways.They have been shown to support the proliferation, growth, anddifferentiation of pluripotential hematopoietic stem cells into vastnumbers of progenitors comprising diverse cellular lineages making up acomplex immune system. Proper and balanced interactions between thecellular components are necessary for a healthy immune response. Thedifferent cellular lineages often respond in a different manner whenlymphokines are administered in conjunction with other agents.

Cell lineages especially important to the immune response include twoclasses of lymphocytes: B-cells, which can produce and secreteimmunoglobulins (proteins with the capability of recognizing and bindingto foreign matter to effect its removal), and T-cells of various subsetsthat secrete lymphokines and induce or suppress the B-cells and variousother cells (including other T-cells) making up the immune network.These lymphocytes interact with many other cell types.

Another important cell lineage is the mast cell (which has not beenpositively identified in all mammalian species), which is agranule-containing connective tissue cell located proximal tocapillaries throughout the body. These cells are found in especiallyhigh concentrations in the lungs, skin, and gastrointestinal andgenitourinary tracts. Mast cells play a central role in allergy-relateddisorders, particularly anaphylaxis as follows: when selected antigenscrosslink one class of immunoglobulins bound to receptors on the mastcell surface, the mast cell degranulates and releases mediators, e.g.,histamine, serotonin, heparin, and prostaglandins, which cause allergicreactions, e.g., anaphylaxis.

IL-12 plays a critical role in cell-mediated immunity (Gately et al.(1998) Annu. Rev. Immunol, 16:495-521;Trinchieri (1998) Adv. Immunol;70:83-243; and Trinchieri (1995) Annu. Rev. Immunol. 13:251-276). Itsactivities are triggered through a high-affinity receptor complex thatgathers two closely related subunits, IL-12Rβ1 and β2 (Chua, et al.(1995) J. Immunol. 155:4296-4294; and Preskey et al. (1996b) Proc. Natl.Acad. sci. USA 93:14002-14007). The p35 subunit has been suggested tobind to a second soluble cytokine receptor called EBI3 (Devergne et al.(1997) Proc. Natl. Acad. Sci. USA 94:12041-12046). As yet no biologicalactivity has been reported for the p35-EBI3 pair, however, pairings ofIL-12 subunits or IL-12-like subunits with other cytokines may provideinformation about cell-mediated immunity, e.g. T-cell regulation.Furthermore, the discovery of receptors or receptor subunits for theseheteromeric cytokines will also provide information regarding immuneregulation.

Research to better understand and treat various immune disorders hasbeen hampered by the general inability to maintain cells of the immunesystem in vitro. Immunologists have discovered that culturing thesecells can be accomplished through the use of T-cell and other cellsupernatants, which contain various growth factors, including many ofthe lymphokines.

From the foregoing, it is evident that the discovery and development ofnew lymphokines and their related receptors or receptor subunits, e.g.,related to the IL-6/IL-12 cytokine family could contribute to newtherapies for a wide range of degenerative or abnormal conditions, whichdirectly or indirectly involve the immune system and/or hematopoieticcells. In particular, the discovery and development of lymphokines whichenhance or potentiate the beneficial activities of known lymphokineswould be highly advantageous. The present invention provides newinterleukin compositions, receptor subunits, and related compounds, andmethods for their use.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the comparison between SEQ ID NO: 2 and the IL-D80 variantpolypeptide of SEQ ID NO: 6.

FIG. 2 shows a comparison of rodent IL-D80 (SEQ ID NO: 4) and variantrodent IL-D80 (SEQ ID NO: 8) polypeptide sequences.

FIG. 3 shows a comparison of human IL-D80 (SEQ ID NO: 6) and rodent,e.g., mouse IL-D80 (SEQ ID NO: 8)

SUMMARY OF THE INVENTION

The present invention is directed to mammalian, e.g., rodent, canine,feline, primate, interleukin numbered DNAX 80 (IL-D80; p28) and itsbiological activities. The present invention is also based upon thediscovery of the association of IL-D80 with the IL-12p40-like molecule,EBI3, and the binding of this composite cytokine to an IL-12Rβ2 subunithomologue known as WSX-1/TCCR. The IL-D80/EBI3 composite cytokine isalso known as IL-27. It includes nucleic acids coding for polypeptidesthemselves and methods for their production and use. The nucleic acidsof the invention are characterized, in part, by their homology tocomplementary DNA (cDNA) sequences disclosed herein, and/or byfunctional assays for growth factor- or cytokine-like activities, e.g.,IL-6/IL-12 family of cytokines (see Thomson (1998) The Cytokine Handbook3d ed., Academic Press, San Diego), applied to the polypeptides, whichare typically encoded by these nucleic acids. Methods for modulating orintervening in the control of a growth factor dependent physiology or animmune response are provided.

The present invention is based, in part, upon the discovery of newcytokine sequences exhibiting significant sequence and structuralsimilarity to the IL-6/IL12 family of cytokines. In particular, itprovides primate, e.g., human, and rodent, e.g., mouse, sequences.Functional equivalents exhibiting significant sequence homology will beavailable from other mammalian, e.g., cow, horse, and rat, mouse, andnon-mammalian species.

In various protein embodiments, the invention provides: a substantiallypure or recombinant IL-D80 polypeptide exhibiting identity over a lengthof at least about 12 amino acids to SEQ ID NO: 2, 4, 6, or 8; a naturalsequence IL-D80 of SEQ ID NO: 2, 4, 6, or 8; and a fusion proteincomprising IL-D80 sequence of SEQ ID NO: 2, 4, 6, or 8. In certainembodiments, the segment of identity is at least about 14, 17, or 19amino acids.

In other embodiments, the IL-D80 comprises a mature sequence comprisingthe sequences from SEQ ID NO:2, 4, 6, or 8; or exhibits apost-translational modification pattern distinct from natural IL-D80; orthe polypeptide: is from a warm blooded animal selected from a mammal,including a primate; comprises at least one polypeptide segment of SEQID NO: 2, 4, 6, or 8; exhibits a plurality of amino acid residuefragments; is a natural allelic variant of IL-D80; has a length at leastabout 30 amino acids; exhibits at least two non-overlapping epitopeswhich are specific for a primate IL-D80; exhibits sequence identity overa length of at least about 20 amino acids to primate IL-D80; isglycosylated; has a molecular weight of at least 10 kD with naturalglycosylation; is a synthetic polypeptide; is attached to a solidsubstrate; is conjugated to another chemical moiety; is a 5-fold or lesssubstitution from natural sequence; or is a deletion or insertionvariant from a natural sequence. Preferred embodiments include acomposition comprising: a sterile IL-D80 polypeptide; or the IL-D80polypeptide and a carrier, wherein the carrier is: an aqueous compound,including water, saline, and/or buffer; and/or formulated for oral,rectal, nasal, topical, or parenteral administration. In fusion proteinembodiments, the protein can have: mature polypeptide sequence from SEQID NO:2, 4, 6, or 8; a detection or purification tag, including a FLAG,His6, or Ig sequence; and/or sequence of another cytokine or chemokine,including an IL-12.

Kit embodiments include those with an IL-D80 polypeptide, and: acompartment comprising the polypeptide; and/or instructions for use ordisposal of reagents in the kit.

In binding compound embodiments, the compound may have an antigenbinding site from an antibody, which specifically binds to a naturalIL-D80 polypeptide, wherein: the IL-D80 is a primate protein; thebinding compound is an Fv, Fab, or Fab2 fragment; the binding compoundis conjugated to another chemical moiety; or the antibody: is raisedagainst a peptide sequence of a mature polypeptide portion from SEQ IDNO:2, 4, 6, or 8; is raised against a mature IL-D80; is raised to apurified primate IL-D80; is immunoselected; is a polyclonal antibody;binds to a denatured IL-D80; exhibits a Kd of at least 30 μM; isattached to a solid substrate, including a bead or plastic membrane; isin a sterile composition; or is detectably labeled, including aradioactive or fluorescent label. Kits containing binding compoundsinclude those with: a compartment comprising the binding compound;and/or instructions for use or disposal of reagents in the kit. Oftenthe kit is capable of making a qualitative or quantitative analysis.Preferred compositions will comprise: a sterile binding compound; or thebinding compound and a carrier, wherein the carrier is: an aqueouscompound, including water, saline, and/or buffer; and/or formulated fororal, rectal, nasal, topical, or parenteral administration.

Nucleic acid embodiments include an isolated or recombinant nucleic acidencoding an IL-D80 polypeptide or fusion protein, wherein: the IL-D80 isfrom a primate; and/or the nucleic acid: encodes an antigenic peptidesequence of SEQ ID NO:2, 4, 6, or 8; encodes a plurality of antigenicpeptide sequences of SEQ ID NO:2, 4, 6, or 8; exhibits identity to anatural cDNA encoding the segment; is an expression vector; furthercomprises an origin of replication; is from a natural source; comprisesa detectable label; comprises synthetic nucleotide sequence; is lessthan 6 kb, preferably less than 3 kb; is from a primate, including ahuman; comprises a natural full length coding sequence; is ahybridization probe for a gene encoding the IL-D80; or is a PCR primer,PCR product, or mutagenesis primer. The invention also provides a cell,tissue, or organ comprising such a recombinant nucleic acid, andpreferably the cell will be: a prokaryotic cell; a eukaryotic cell; abacterial cell; a yeast cell; an insect cell; a mammalian cell; a mousecell; a primate cell; or a human cell.

Kit embodiments include those with such nucleic acids, and: acompartment comprising the nucleic acid; a compartment furthercomprising the IL-D80 protein or polypeptide; and/or instructions foruse or disposal of reagents in the kit. Typically, the kit is capable ofmaking a qualitative or quantitative analysis.

In certain embodiments, the nucleic acid: hybridizes under washconditions of 30° C. and less than 2M salt, or of 45° C. and/or 500 mMsalt, or 55° C. and/or 150 mM salt, to SEQ ID NO: 1, 3, 5, or 7; orexhibits identity over a stretch of at least about 30, 55, or75nucleotides, to a primate IL-D80.

The invention embraces a method of modulating physiology or developmentof a cell or tissue culture cells comprising contacting the cell with anagonist or antagonist of a primate IL-D80. The method may be where: thecontacting is in combination with an agonist or antagonist of IL-12; orthe contacting is with an antagonist, including a binding compositioncomprising an antibody binding site which specifically binds an IL-D80.

The invention further provides a composite cytokine (IL-27) comprising aplurality of segments of SEQ ID NO:2, 4, 6, or 8 and SEQ ID NO:10. Alsoencompassed is an isolated or recombinant polynucleotide encoding thecomposite cytokine of said composite cytokine. Further provided is areceptor subunit:ligand composition comprising a plurality ofpolypeptide segments of SEQ ID NO:2, 4, 6, or 8, SEQ ID NO:10, and SEQID NO:12.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

All references cited herein are incorporated herein by reference to thesame extent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

I. General

The present invention provides amino acid sequences and DNA sequencesencoding various mammalian proteins, which are cytokines, e.g., whichare secreted molecules which can mediate a signal between immune orother cells. See, e.g., Paul (1997) Fundamental Immunology (3d ed.)Raven Press, N.Y. The full length cytokines, and fragments, orantagonists will be useful in physiological modulation of cellsexpressing a receptor. It is likely that IL-D80 or IL-27 has eitherstimulatory or inhibitory effects on hematopoietic cells, including,e.g., lymphoid cells, such as T-cells, B-cells, natural killer (NK)cells, macrophages, dendritic cells, hematopoietic progenitors, etc. Inparticular, the IL-27 composite cytokine may play a role ininflammation, including, but not limited to ulcerative colitis,arthritis, etc. The proteins will also be useful as antigens, e.g.,immunogens, for raising antibodies to various epitopes on the protein,both linear and conformational epitopes.

A cDNA encoding IL-D80 was identified from various primate, e.g., human,sequences of BACs of Chromosome 16. See, e.g., CIT987SK-A-575C2, andCIT987SK-A-761H5. The molecule was designated huIL-D80. A human EST hasbeen identified and described, human EST AI085007. A mouse EST AA266872has also been identified and described.

The primate, e.g., human, gene will encode a small soluble cytokine-likeprotein of about 243 amino acids . See SEQ. ID. NOs: 1, 2, 5, and 6.Exon boundaries are likely to correspond to about 219/220; 393/394;492/493; and 551/552 of SEQ ID NO:1. Coding segments corresponding tothose boundaries are particularly interesting. Translated amino acidsequence, which is encoded by nucleotides 193 to 918 of SEQ ID NO:1, isshown in SEQ ID NO: 2.

A predicted signal cleavage site may exist between about residues 25-30of SEQ ID NO: 2; helix A is predicted to run from about residues 33-38to about residues 54-59 of SEQ ID NO: 2; helix B is predicted to runfrom about residues 85-90 to about residues 111-116 of SEQ ID NO: 2;helix C is predicted to run from about residues 121-126 to aboutresidues 154-159 of SEQ ID NO: 2; and helix D is predicted to run fromabout residues 201-206 to about residues 228-233 of SEQ ID NO: 2.

SEQ ID NO: 5 shows a variant of IL-D80 and SEQ ID NO: 6 is the encodedpolypeptide. FIG. 1 shows the comparison between SEQ ID NO: 2 and theIL-D80 variant polypeptide of SEQ ID NO: 6. Structural motifs are asindicated above with the appropriate change in residue positions.

The corresponding rodent polynucleotide sequence of IL-D80 is shown inSEQ ID NO: 3. Exon boundaries are likely to run from about 198/199;360/361; 459/460; and 618/619. The predicted polypeptide sequence, whichruns from about nucleotide 199 to 891 of SEQ ID NO: 3, is shown in SEQID NO: 4.The predicted signal cleavage site runs from about residue16-21 of SEQ ID NO: 4; helix A is predicted to run from about residue21-26 to about residue 41-46; helix B is predicted to run from aboutresidue 72-77 to about residue 101-106; helix C is predicted to run fromabout residue 108-133 to about residue 141-146; and helix D is predictedto run from about residue 185-190 to about residue 211-215. All postionsrefer to SEQ ID NO: 4. A variant rodent IL-D80 polynucleotide sequenceis shown in SEQ ID NO: 7 and the predicted polypeptide sequence is shownin SEQ ID NO: 8. A comparison of rodent IL-D80 (SEQ ID NO: 4) andvariant rodent IL-D80 (SEQ ID NO: 8) polypeptide sequences is shown inFIG. 2.

IL-D80 exhibits structural motifs characteristic of a member of the longchain cytokines belonging to the IL-6/IL-12 family of cytokines. Thestructural homology of IL-D80 to related cytokine proteins suggestsrelated function of this molecule.

The IL-D80 cDNA sequences mature proteins with calculated molecular massof 24.5 and 23.6 kDa. No N-glycosylation sites are found in hIL-D80, butseveral O-glycosylation sites are predicted. Murine IL-D80 contains onepotential N-glycosylation site (N85). Transient expression of mp28 inthe presence or absence of tunicamycin indicated that mp28 is indeedN-linked glycosylated. Both human and mouse IL-D80 display an unusualsequence insertion in the predicted loop region between helix C and D.In hIL-D80, the C-D loop contains a stretch of 13 glutamic acidresidues; mp28 displays 14 negatively charged residues in this region,interrupted by one lysine residue. This highly charged sequence has notbeen observed in any other helical cytokine and most likely will affectthe biophysical properties of the protein in solution. Overall, humanand mouse IL-D80 are 74% identical.

Comparison of the sequences will also provide an evolutionary tree. Thiscan be generated, e.g., using the TreeView program in combination withthe ClustalX analysis software program. See Thompson, et al. Nuc. AcidsRes. 25:4876-4882; and TreeView, Page, IBLS, University of Glasgow,e-mail rpage@bio.gla.ac.uk;http://taxonomy.zoology.gla.ac.uk.rod.treeview.html.

Co-transfection of human Epstein-Barr virus-induced gene 3 (EBI3;GenBank NM005755; Devergne, et al. (1996) J. Virol. 70:1143-1153; SEQ IDNOs: 9 and 10) cDNA and human IL-D80 cDNA leads to enhanced secretion ofIL-D80. IL-D80 co-immunoprecipitated with EBI3, and conversely, EBI3co-immunoprecipitated with IL-D80. This indicates that these twoproteins form a composite factor that either itself has biologicalfunctions (that neither protein has on its own) or EBI3 is used as ashuttle to release IL-D80 in the supernatant. Of note, EBI3 is alsoexpressed in vivo by activated antigen presenting cells (APCs) and atvery high levels by placental syncytiotrophoblasts. The presentinvention provides the first evidence that the IL-80D/EBI3 compositecytokine (IL-27) binds to an IL-12R-like subunit, WSX-1/TCCR (See, e.g.,GenBank AF265242; Chen, et al. (2000) Nature 407:916-920; SEQ ID NO: 11and 12).

Biologically, IL-27 is produced by antigen presenting cells (APCs). Incontrast to other similar heterodimers made by APCs, i.e., IL-12(p35+p40) and IL-23 (p19+p40), kinetic analysis of IL-27 showed thatthis composite cytokine is produced earlier in activation of APCs. Thus,IL-27 can be a potent adjuvant of a Th1 response.

The primary activity of IL-27 triggers rapid clonal expansion of antigenspecific for naive human and mouse CD4+ T cells. Moreover, it promotesTh1 polarization and IFN-γ production of naïve CD4+ T cells.Mechanistically, these naive T cells are primed to respond to IL-27 bythe production of this composite cytokine by the APCs which interactwith these cells. These activities of IL-27 are dependent onsimultaneous T cell receptor activation and occur in synergy with IL-12.

IL-D80 or IL-27 agonists, or antagonists, may also act as functional orreceptor antagonists. Thus, IL-D80, IL-27, WSX-1/TCCR, or itsantagonists, may be useful in the treatment of abnormal medicalconditions, including immune disorders, e.g., T cell immunedeficiencies, inflammation, or tissue rejection, or in cardiovascular orneurophysiological conditions.

The natural antigens are capable of mediating various biochemicalresponses which lead to biological or physiological responses in targetcells. The preferred embodiment characterized herein is from human, butother primate, or other species counterparts exist in nature. Additionalsequences for proteins in other mammalian species, e.g., primates,canines, felines, and rodents, should also be available, particularlythe domestic animal species. See below. The descriptions below aredirected, for exemplary purposes, to a human IL-D80 or IL-27, but arelikewise applicable to related embodiments from other species.

II. Purified IL-D80 or IL-27

Mammlian IL-D80 amino acid sequence, is shown in several embodiments,e.g., SEQ ID NO: 2, 4, 6, or 8. EBI3 amino acid sequence is provided inSEQ ID NO: 10. Other naturally occurring nucleic acids which encode theprotein can be isolated by standard procedures using the providedsequence, e.g., PCR techniques, or by hybridization. These amino acidsequences, provided amino to carboxy, are important in providingsequence information for the cytokine allowing for distinguishing theprotein antigen from other proteins and exemplifying numerous variants.Moreover, the peptide sequences allow preparation of peptides togenerate antibodies to recognize such segments, and nucleotide sequencesallow preparation of oligonucleotide probes, both of which arestrategies for detection or isolation, e.g., cloning, of genes encodingsuch sequences.

As used herein, the term “human soluble IL-D80 or IL-27” shallencompass, when used in a protein context, a protein having amino acidsequence corresponding to a soluble polypeptide shown in SEQ ID NO: 2 or6, or significant fragments thereof. Preferred embodiments comprise aplurality of distinct, e.g., nonoverlapping, segments of the specifiedlength. Typically, the plurality will be at least two, more usually atleast three, and preferably 5, 7, or even more. While the length minimaare provided, longer lengths, of various sizes, may be appropriate,e.g., one of length 7, and two of length 12.

Binding components, e.g., antibodies, typically bind to an IL-D80 orIL-27 with high affinity, e.g., at least about 100 nM, usually betterthan about 30 nM, preferably better than about 10 nM, and morepreferably at better than about 3 nM. Counterpart proteins will be foundin mammalian species other than human, e.g., other primates, ungulates,or rodents. Non-mammalian species should also possess structurally orfunctionally related genes and proteins, e.g., birds or amphibians.

The term “polypeptide” as used herein includes a significant fragment orsegment, and encompasses a stretch of amino acid residues of at leastabout 8 amino acids, generally at least about 12 amino acids, typicallyat least about 16 amino acids, preferably at least about 20 amino acids,and, in particularly preferred embodiments, at least about 30 or moreamino acids, e.g., 35, 40, 45, 50, 60, 75, 100, etc. Such fragments mayhave ends which begin and/or end at virtually all positions, e.g.,beginning at residues 1, 2, 3, etc., and ending at, e.g., 150, 149, 148,etc., in all practical combinations. Particularly interesting peptideshave ends corresponding to structural domain boundaries, e.g., helicesA, B, C, and/or D.

The term “binding composition” refers to molecules that bind withspecificity to IL-D80 or IL-27, e.g., in an antibody-antigeninteraction. The specificity may be more or less inclusive, e.g.,specific to a particular embodiment, or to groups of relatedembodiments, e.g., primate, rodent, etc. It also includes compounds,e.g., proteins, which specifically associate with IL-D80 or IL-27,including in a natural physiologically relevant protein-proteininteraction, either covalent or non-covalent. The molecule may be apolymer, or chemical reagent. A functional analog may be a protein withstructural modifications, or it may be a molecule which has a molecularshape which interacts with the appropriate binding determinants. Thecompounds may serve as agonists or antagonists of a receptor bindinginteraction, see, e.g., Goodman, et al. (eds.) Goodman & Gilman's: ThePharmacological Bases of Therapeutics (current ed.) Pergamon Press.

Substantially pure, e.g., in a protein context, typically means that theprotein is free from other contaminating proteins, nucleic acids, orother biologicals derived from the original source organism. Purity maybe assayed by standard methods, typically by weight, and will ordinarilybe at least about 40% pure, generally at least about 50% pure, often atleast about 60% pure, typically at least about 80% pure, preferably atleast about 90% pure, and in most preferred embodiments, at least about95% pure. Carriers or excipients will often be added.

Solubility of a polypeptide or fragment depends upon the environment andthe polypeptide. Many parameters affect polypeptide solubility,including temperature, electrolyte environment, size and molecularcharacteristics of the polypeptide, and nature of the solvent.Typically, the temperature at which the polypeptide is used ranges fromabout 4° C. to about 65° C. Usually the temperature at use is greaterthan about 18° C. For diagnostic purposes, the temperature will usuallybe about room temperature or warmer, but less than the denaturationtemperature of components in the assay. For therapeutic purposes, thetemperature will usually be body temperature, typically about 37° C. forhumans and mice, though under certain situations the temperature may beraised or lowered in situ or in vitro.

The size and structure of the polypeptide should generally be in asubstantially stable state, and usually not in a denatured state. Thepolypeptide may be associated with other polypeptides in a quaternarystructure, e.g., to confer solubility, or associated with lipids ordetergents.

The solvent and electrolytes will usually be a biologically compatiblebuffer, of a type used for preservation of biological activities, andwill usually approximate a physiological aqueous solvent. Usually thesolvent will have a neutral pH, typically between about 5 and 10, andpreferably about 7.5. On some occasions, one or more detergents will beadded, typically a mild non-denaturing one, e.g., CHS (cholesterylhemisuccinate) or CHAPS (3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), or a low enough concentration as to avoid significantdisruption of structural or physiological properties of the protein. Inother instances, a harsh detergent may be used to effect significantdenaturation.

The above will also be applicable to the IL-D80 or IL-27/EBI3 compositecytokine, where SEQ ID NO: 10 is the polypeptide sequence of EBI3.

III. Physical Variants

This invention also encompasses proteins or peptides having substantialamino acid sequence identity with the amino acid sequence of the IL-D80or IL-27 antigen. The variants include species, polymorphic, or allelicvariants.

Amino acid sequence homology, or sequence identity, is determined byoptimizing residue matches, if necessary, by introducing gaps asrequired. See also Needleham, et al. (1970) J. Mol. Biol. 48:443-453;Sankoff, et al. (1983) Chapter One in Time Warps, String Edits, andMacromolecules: The Theory and Practice of Sequence Comparison,Addison-Wesley, Reading, Mass.; and software packages fromIntelliGenetics, Mountain View, Calif.; and the University of WisconsinGenetics Computer Group, Madison, Wis. Sequence identity changes whenconsidering conservative substitutions as matches. Conservativesubstitutions typically include substitutions within the followinggroups: glycine, alanine; valine, isoleucine, leucine; aspartic acid,glutamic acid; asparagine, glutamine; serine, threonine; lysine,arginine; and phenylalanine, tyrosine. The conservation may apply tobiological features, functional features, or structural features.Homologous amino acid sequences are typically intended to includenatural polymorphic or allelic and interspecies variations of a proteinsequence. Typical homologous proteins or peptides will have from 25-100%identity (if gaps can be introduced), to 50-100% identity (ifconservative substitutions are included) with the amino acid sequence ofthe IL-D80 or IL-27. Identity measures will be at least about 35%,generally at least about 40%, often at least about 50%, typically atleast about 60%, usually at least about 70%, preferably at least about80%, and more preferably at least about 90%.

The isolated IL-D80 or IL-27 DNA can be readily modified by nucleotidesubstitutions, nucleotide deletions, nucleotide insertions, andinversions of short nucleotide stretches. These modifications result innovel DNA sequences which encode these antigens, their derivatives, orproteins having similar physiological, immunogenic, antigenic, or otherfunctional activity. These modified sequences can be used to producemutant antigens or to enhance expression. Enhanced expression mayinvolve gene amplification, increased transcription, increasedtranslation, and other mechanisms. “Mutant IL-D80 or IL-27” encompassesa polypeptide otherwise falling within the sequence identity definitionof the IL-D80 or IL-27 as set forth above, but having an amino acidsequence which differs from that of IL-D80 or IL-27 as normally found innature, whether by way of deletion, substitution, or insertion. Thisgenerally includes proteins having significant identity with a proteinhaving sequence of SEQ ID NO: 2, 4, 6, or 8, or the foregoing inassociation with SEQ ID NO: 10 and as sharing various biologicalactivities, e.g., antigenic or immunogenic, with those sequences, and inpreferred embodiments contain most of the natural full length disclosedsequences. Full length sequences will typically be preferred, thoughtruncated versions will also be useful, likewise, genes or proteinsfound from natural sources are typically most desired. Similar conceptsapply to different IL-D80 or IL-27 proteins, particularly those found invarious warm blooded animals, e.g., mammals and birds. Thesedescriptions are generally meant to encompass many IL-D80 or IL-27proteins, not limited to the particular mammalian embodimentsspecifically discussed.

IL-D80 or IL-27 mutagenesis can also be conducted by making amino acidinsertions or deletions. Substitutions, deletions, insertions, or anycombinations may be generated to arrive at a final construct. Insertionsinclude amino- or carboxy-terminal fusions. Random mutagenesis can beconducted at a target codon and the expressed mutants can then bescreened for the desired activity. Methods for making substitutionmutations at predetermined sites in DNA having a known sequence are wellknown in the art, e.g., by M13 primer mutagenesis or polymerase chainreaction (PCR) techniques. See, e.g., Sambrook, et al. (1989); Ausubel,et al. (1987 and Supplements); and Kunkel, et al. (1987) Methods inEnzymol. 154:367-382. Preferred embodiments include, e.g., 1-fold,2-fold, 3-fold, 5-fold, 7-fold, etc., preferably conservativesubstitutions at the nucleotide or amino acid levels. Preferably thesubstitutions will be away from the conserved cysteines, and often willbe in the regions away from the helical structural domains. Suchvariants may be useful to produce specific antibodies, and often willshare many or all biological properties.

The present invention also provides recombinant proteins, e.g.,heterologous fusion proteins using segments from these proteins. Aheterologous fusion protein is a fusion of proteins or segments whichare naturally not normally fused in the same manner. A similar conceptapplies to heterologous nucleic acid sequences.

In addition, new constructs may be made from combining similarfunctional domains from other proteins. For example, target-binding orother segments may be “swapped” between different new fusionpolypeptides or fragments. See, e.g., Cunningham, et al. (1989) Science243:1330-1336; and O'Dowd, et al. (1988) J. Biol. Chem. 263:15985-15992.

The phosphoramidite method described by Beaucage and Carruthers (1981)Tetra. Letts. 22:1859-1862, will produce suitable synthetic DNAfragments. A double stranded fragment will often be obtained either bysynthesizing the complementary strand and annealing the strand togetherunder appropriate conditions or by adding the complementary strand usingDNA polymerase with an appropriate primer sequence, e.g., PCRtechniques.

Structural analysis can be applied to this gene, in comparison to theIL-12 family of cytokines. In particular, β-sheet and α-helix residuescan be determined using, e.g., RASMOL program, see Bazan, et al. (1996)Nature 379:591; Lodi, et al. (1994) Science 263:1762-1766; Sayle andMilner-White (1995) Trends in Biol. Sci. 20:374-376; and Gronenberg, etal. (1991) Protein Engineering 4:263-269. Preferred residues forsubstitutions include the surface exposed residues which would bepredicted to interact with receptor. Other residues which shouldconserve function will be conservative substitutions, particularly atpositions far from the surface exposed residues.

The above will also be applicable for the IL-D80 or IL-27 (i.e.,IL-D80+EBI3) composite cytokine where SEQ ID NO: 10 is the polypeptidesequence of EBI3.

IV. Functional Variants

The blocking of physiological response to IL-D80 or the IL-27 compositecytokine may result from the competitive inhibition of binding of theligand to its receptor.

In vitro assays of the present invention will often use isolatedprotein, soluble fragments comprising receptor binding segments of theseproteins, or fragments attached to solid phase substrates. These assayswill also allow for the diagnostic determination of the effects ofeither binding segment mutations and modifications, or cytokinemutations and modifications, e.g., IL-D80 or IL-27 analogs.

This invention also contemplates the use of competitive drug screeningassays, e.g., where neutralizing antibodies to the cytokine, or receptorbinding fragments compete with a test compound.

“Derivatives” of IL-D80 or IL-27 antigens include amino acid sequencemutants from naturally occurring forms, glycosylation variants, andcovalent or aggregate conjugates with other chemical moieties, e.g.,PEGylation. Covalent derivatives can be prepared by linkage offunctionalities to groups which are found in IL-D80 or IL-27 amino acidside chains or at the N- or C-termini, e.g., by standard means. See,e.g., Lundblad and Noyes (1988) Chemical Reagents for ProteinModification, vols. 1-2, CRC Press, Inc., Boca Raton, Fla.; Hugli (ed.1989) Techniques in Protein Chemistry, Academic Press, San Diego,Calif.; and Wong (1991) Chemistry of Protein Conjugation and CrossLinking, CRC Press, Boca Raton, Fla.

In particular, glycosylation alterations are included, e.g., made bymodifying the glycosylation patterns of a polypeptide during itssynthesis and processing, or in further processing steps. See, e.g.,Elbein (1987) Ann. Rev. Biochem. 56:497-534. Also embraced are versionsof the peptides with the same primary amino acid sequence which haveother minor modifications, including phosphorylated amino acid residues,e.g., phosphotyrosine, phosphoserine, or phosphothreonine.

Fusion polypeptides between IL-D80 or IL-27 and other homologous orheterologous proteins are also provided. Many cytokine receptors orother surface proteins are multimeric, e.g., homodimeric entities, and arepeat construct may have various advantages, including lessenedsusceptibility to proteolytic cleavage. Typical examples are fusions ofa reporter polypeptide, e.g., luciferase, with a segment or domain of aprotein, e.g., a receptor-binding segment, so that the presence orlocation of the fused ligand may be easily determined. See, e.g., Dull,et al., U.S. Pat. No. 4,859,609. Other gene fusion partners includebacterial β-galactosidase, trpE, Protein A, β-lactamase, alpha amylase,alcohol dehydrogenase, yeast alpha mating factor, and detection orpurification tags such as a FLAG sequence of His6 sequence. See, e.g.,Godowski, et al. (1988) Science 241:812-816.

Fusion peptides will typically be made by either recombinant nucleicacid methods or by synthetic polypeptide methods. Techniques for nucleicacid manipulation and expression are described generally, e.g., inSambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.),vols. 1-3, Cold Spring Harbor Laboratory; and Ausubel, et al. (eds.1993) Current Protocols in Molecular Biology, Greene and Wiley, NY.Techniques for synthesis of polypeptides are described, e.g., inMerrifield (1963) J. Amer. Chem. Soc. 85:2149-2156; Merrifield (1986)Science 232: 341-347; Atherton, et al. (1989) Solid Phase PeptideSynthesis: A Practical Approach, IRL Press, Oxford; and Grant (1992)Synthetic Peptides: A User's Guide, W. H. Freeman, NY. Refolding methodsmay be applicable to synthetic proteins.

This invention also contemplates the use of derivatives of IL-D80 orIL-27 proteins other than variations in amino acid sequence orglycosylation. Such derivatives may involve covalent or aggregativeassociation with chemical moieties or protein carriers. Covalent oraggregative derivatives will be useful as immunogens, as reagents inimmunoassays, or in purification methods such as for affinitypurification of binding partners, e.g., other antigens. An IL-D80 orIL-27 can be immobilized by covalent bonding to a solid support such ascyanogen bromide-activated SEPHAROSE, by methods which are well known inthe art, or adsorbed onto polyolefin surfaces, with or withoutglutaraldehyde cross-linking, for use in the assay or purification ofanti-IL-D80 or IL-27 antibodies or an alternative binding composition.The IL-D80 or IL-27 proteins can also be labeled with a detectablegroup, e.g., for use in diagnostic assays. Purification of IL-D80 orIL-27 may be effected by an immobilized antibody or complementarybinding partner, e.g., binding portion of a receptor.

A solubilized IL-D80 or IL-27, or fragments of this invention can beused as an immunogen for the production of antisera or antibodiesspecific for binding. Purified antigen can be used to screen monoclonalantibodies or antigen-binding fragments, encompassing antigen bindingfragments of natural antibodies, e.g., Fab, Fab′, F(ab)₂, etc. PurifiedIL-D80 or IL-27 antigens can also be used as a reagent to detectantibodies generated in response to the presence of elevated levels ofthe cytokine, which may be diagnostic of an abnormal or specificphysiological or disease condition. This invention contemplatesantibodies raised against amino acid sequences encoded by nucleotidesequence shown in SEQ ID NO: 1, 3, 5, or 7, or fragments of proteinscontaining it. Also contemplated are sequences encoding the IL-D80 orIL-27 cytokines, or fragments thereof. In particular, this inventioncontemplates antibodies having binding affinity to or being raisedagainst specific domains, e.g., helices A, B, C, or D.

The present invention contemplates the isolation of additional closelyrelated species variants. Southern and Northern blot analysis willestablish that similar genetic entities exist in other mammals. It islikely that IL-D80 or IL-27s are widespread in species variants, e.g.,rodents, lagomorphs, carnivores, artiodactyla, perissodactyla, andprimates.

The invention also provides means to isolate a group of related antigensdisplaying both distinctness and similarities in structure, expression,and function. Elucidation of many of the physiological effects of themolecules will be greatly accelerated by the isolation andcharacterization of additional distinct species or polymorphic variantsof them. In particular, the present invention provides useful probes foridentifying additional homologous genetic entities in different species.

The isolated genes will allow transformation of cells lacking expressionof an IL-D80 or IL-27, e.g., either species types or cells which lackcorresponding proteins and exhibit negative background activity. Thisshould allow analysis of the function of IL-D80 or IL-27 in comparisonto untransformed control cells.

Dissection of critical structural elements which effect the variousphysiological functions mediated through these antigens is possibleusing standard techniques of modern molecular biology, particularly incomparing members of the related class. See, e.g., the homolog-scanningmutagenesis technique described in Cunningham, et al. (1989) Science243:1339-1336; and approaches used in O'Dowd, et al. (1988) J. Biol.Chem. 263:15985-15992; and Lechleiter, et al. (1990) EMBO J.9:4381-4390.

Intracellular functions would probably involve receptor signaling.However, protein internalization may occur under certain circumstances,and interaction between intracellular components and cytokine may occur.Specific segments of interaction of IL-D80 or IL-27 with interactingcomponents may be identified by mutagenesis or direct biochemical means,e.g., cross-linking or affinity methods. Structural analysis bycrystallographic or other physical methods will also be applicable.Further investigation of the mechanism of signal transduction willinclude study of associated components which may be isolatable byaffinity methods or by genetic means, e.g., complementation analysis ofmutants.

Further study of the expression and control of IL-D80 or IL-27 will bepursued. The controlling elements associated with the antigens shouldexhibit differential physiological, developmental, tissue specific, orother expression patterns. Upstream or downstream genetic regions, e.g.,control elements, are of interest.

Structural studies of the IL-D80 or IL-27 antigens will lead to designof new antigens, particularly analogs exhibiting agonist or antagonistproperties on the molecule. This can be combined with previouslydescribed screening methods to isolate antigens exhibiting desiredspectra of activities.

V. Antibodies

Antibodies can be raised to various epitopes of the IL-D80 or IL-27proteins, including species, polymorphic, or allelic variants, andfragments thereof, both in their naturally occurring forms and in theirrecombinant forms. Additionally, antibodies can be raised to IL-D80 orIL-27 s in either their active forms or in their inactive forms,including native or denatured versions. Anti-idiotypic antibodies arealso contemplated.

Antibodies, including binding fragments and single chain versions,against predetermined fragments of the antigens can be raised byimmunization of animals with conjugates of the fragments withimmunogenic proteins. Monoclonal antibodies are prepared from cellssecreting the desired antibody. These antibodies can be screened forbinding to normal or defective IL-D80 or IL-27s, or screened foragonistic or antagonistic activity, e.g., mediated through a receptor.Antibodies may be agonistic or antagonistic, e.g., by stericallyblocking binding to a receptor. These monoclonal antibodies will usuallybind with at least a K_(D) of about 1 mM, more usually at least about300 μM, typically at least about 100 μM, more typically at least about30 μM, preferably at least about 10 μM, and more preferably at leastabout 3 μM or better.

An IL-D80 or IL-27 protein that specifically binds to or that isspecifically immunoreactive with an antibody generated against a definedimmunogen, such as an immunogen consisting of the amino acid sequence ofSEQ ID NO: 2, 4, 6, or 8, or any of the foregoing in association withSEQ ID NO: 10, is typically determined in an immunoassay. Theimmunoassay typically uses a polyclonal antiserum which was raised,e.g., to a polypeptide of SEQ ID NO: 2, 4, 6, or 8, or any of theforegoing in association with SEQ ID NO: 10. This antiserum is selectedto have low crossreactivity against other IL12 family members, e.g.,human or rodent IL-12, preferably from the same species, and any suchcrossreactivity is removed by immunoabsorption prior to use in theimmunoassay.

In order to produce antisera for use in an immunoassay, the protein ofSEQ ID NO: 2, 4, 6, or 8, or the foregoing in association with SEQ IDNO: 10, or a combination thereof, is isolated as described herein. Forexample, recombinant protein may be produced in a mammalian cell line.An appropriate host, e.g., an inbred strain of mice such as Balb/c, isimmunized with the selected protein, typically using a standardadjuvant, such as Freund's adjuvant, and a standard mouse immunizationprotocol (see Harlow and Lane, supra). Alternatively, a syntheticpeptide derived from the sequences disclosed herein and conjugated to acarrier protein can be used an immunogen. Polyclonal sera are collectedand titered against the immunogen protein in an immunoassay, e.g., asolid phase immunoassay with the immunogen immobilized on a solidsupport. Polyclonal antisera with a titer of 10⁴ or greater are selectedand tested for their cross reactivity against other IL-12 familymembers, e.g., rodent IL-12, using a competitive binding immunoassaysuch as the one described in Harlow and Lane, supra, at pages 570-573.Preferably at least one other IL-12 family member is used in thisdetermination in conjunction with, e.g., the primate IL-12. The IL-12family members can be produced as recombinant proteins and isolatedusing standard molecular biology and protein chemistry techniques asdescribed herein.

Immunoassays in the competitive binding format can be used for thecrossreactivity determinations. For example, the protein of SEQ ID NO: 2or 6 can be immobilized to a solid support. Proteins added to the assaycompete with the binding of the antisera to the immobilized antigen. Theability of the above proteins to compete with the binding of theantisera to the immobilized protein is compared to the protein of SEQ IDNO: 2 or 6. Similarly, the composite cytokine of SEQ ID NO: 2 or 6 inassociation with SEQ ID NO: 10 can be used. The percent crossreactivityfor the above proteins is calculated, using standard calculations. Thoseantisera with less than 10% crossreactivity with each of the proteinslisted above are selected and pooled. The cross-reacting antibodies arethen removed from the pooled antisera by immunoabsorption with theabove-listed proteins.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein tothe immunogen protein (e.g., the IL-12 like protein of SEQ ID NO: 2, 4,6, or 8, or any of the foregoing in association with SEQ ID NO: 10). Inorder to make this comparison, the two proteins are each assayed at awide range of concentrations and the amount of each protein required toinhibit 50% of the binding of the antisera to the immobilized protein isdetermined. If the amount of the second protein required is less thantwice the amount of the protein of the selected protein or proteins thatis required, then the second protein is said to specifically bind to anantibody generated to the immunogen.

The antibodies of this invention can also be useful in diagnosticapplications. As capture or non-neutralizing antibodies, they can bescreened for ability to bind to the antigens without inhibiting bindingto a receptor. As neutralizing antibodies, they can be useful incompetitive binding assays. They will also be useful in detecting orquantifying IL-D80 or IL-27 protein or its receptors, e.g., WSX-1/TCCR(SEQ ID NO: 12). See, e.g., Chan (ed. 1987) Immunology: A PracticalGuide, Academic Press, Orlando, Fla.; Price and Newman (eds. 1991)Principles and Practice of Immunoassay, Stockton Press, N.Y.; and Ngo(ed. 1988) Nonisotopic Immunoassay, Plenum Press, N.Y. Crossabsorptions, depletions, or other means will provide preparations ofdefined selectivity, e.g., unique or shared species specificities. Thesemay be the basis for tests which will identify various groups ofantigens.

Further, the antibodies, including antigen binding fragments, of thisinvention can be potent antagonists that bind to the antigen and inhibitfunctional binding, e.g., to a receptor which may elicit a biologicalresponse. They also can be useful as non-neutralizing antibodies and canbe coupled to toxins or radionuclides so that when the antibody binds toantigen, a cell expressing it, e.g., on its surface, is killed. Further,these antibodies can be conjugated to drugs or other therapeutic agents,either directly or indirectly by means of a linker, and may effect drugtargeting.

Antigen fragments may be joined to other materials, particularlypolypeptides, as fused or covalently joined polypeptides to be used asimmunogens. An antigen and its fragments may be fused or covalentlylinked to a variety of immunogens, such as keyhole limpet hemocyanin,bovine serum albumin, tetanus toxoid, etc. See Microbiology, HoeberMedical Division, Harper and Row, 1969; Landsteiner (1962) Specificityof Serological Reactions, Dover Publications, New York; Williams, et al.(1967) Methods in Immunology and Immunochemistry, vol. 1, AcademicPress, New York; and Harlow and Lane (1988) Antibodies: A LaboratoryManual, CSH Press, NY, for descriptions of methods of preparingpolyclonal antisera.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious mammalian hosts, such as mice, rodents, primates, humans, etc.Description of techniques for preparing such monoclonal antibodies maybe found in, e.g., Stites, et al. (eds.) Basic and Clinical Immunology(4th ed.), Lange Medical Publications, Los Altos, Calif., and referencescited therein; Harlow and Lane (1988) Antibodies: A Laboratory Manual,CSH Press; Goding (1986) Monoclonal Antibodies: Principles and Practice(2d ed.), Academic Press, New York; and particularly in Kohler andMilstein (1975) Nature 256:495-497, which discusses one method ofgenerating monoclonal antibodies.

Other suitable techniques involve in vitro exposure of lymphocytes tothe antigenic polypeptides or alternatively to selection of libraries ofantibodies in phage or similar vectors. See, Huse, et al. (1989)“Generation of a Large Combinatorial Library of the ImmunoglobulinRepertoire in Phage Lambda,” Science 246:1275-1281; and Ward, et al.(1989) Nature 341:544-546. The polypeptides and antibodies of thepresent invention may be used with or without modification, includingchimeric or humanized antibodies. Frequently, the polypeptides andantibodies will be labeled by joining, either covalently ornon-covalently, a substance which provides for a detectable signal. Awide variety of labels and conjugation techniques are known and arereported extensively in both the scientific and patent literature.Suitable labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent moieties, chemiluminescent moieties, magneticparticles, and the like. Patents, teaching the use of such labelsinclude U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulinsmay be produced, see Cabilly, U.S. Pat. No. 4,816,567; Moore, et al.,U.S. Pat. No. 4,642,334; and Queen, et al. (1989) Proc. Nat'l Acad. Sci.USA 86:10029-10033.

The antibodies of this invention can also be used for affinitychromatography in isolating the protein. Columns can be prepared wherethe antibodies are linked to a solid support. See, e.g., Wilchek et al.(1984) Meth. Enzymol. 104:3-55. The converse may be used to purifyantibodies.

Antibodies raised against each IL-D80 or IL-27 will also be useful toraise anti-idiotypic antibodies. These will be useful in detecting ordiagnosing various immunological conditions related to expression of therespective antigens.

VI. Nucleic Acids

The described peptide sequences and the related reagents are useful indetecting, isolating, or identifying a DNA clone encoding IL-D80 orIL-27, e.g., from a natural source. Typically, it will be useful inisolating a gene from mammal, and similar procedures will be applied toisolate genes from other species, e.g., warm blooded animals, such asbirds and mammals. Cross hybridization will allow isolation of IL-D80 orIL-27 from the same, e.g., polymorphic variants, or other species. Anumber of different approaches will be available to successfully isolatea suitable nucleic acid clone.

The purified protein or defined peptides are useful for generatingantibodies by standard methods, as described above. Synthetic peptidesor purified protein can be presented to an immune system to generatemonoclonal or polyclonal antibodies. See, e.g., Coligan (1991) CurrentProtocols in Immunology Wiley/Greene; and Harlow and Lane (1989)Antibodies: A Laboratory Manual, Cold Spring Harbor Press.

For example, the specific binding composition could be used forscreening of an expression library made from a cell line which expressesan IL-D80 or IL-27. Screening of intracellular expression can beperformed by various staining or immunofluorescence procedures. Bindingcompositions could be used to affinity purify or sort out cellsexpressing a surface fusion protein.

The peptide segments can also be used to predict appropriateoligonucleotides to screen a library. The genetic code can be used toselect appropriate oligonucleotides useful as probes for screening. See,e.g., SEQ ID NO: 1, 3, 5, or 7, or any of the foregoing in addition toSEQ ID NO: 9. In combination with polymerase chain reaction (PCR)techniques, synthetic oligonucleotides will be useful in selectingcorrect clones from a library. Complementary sequences will also be usedas probes, primers, or antisense strands. Various fragments should beparticularly useful, e.g., coupled with anchored vector or poly-Acomplementary PCR techniques or with complementary DNA of otherpeptides.

This invention contemplates use of isolated DNA or fragments to encodean antigenic or biologically active corresponding IL-D80 or IL-27polypeptide, particularly lacking the portion coding the untranslated 5′portion of the described sequence. In addition, this invention coversisolated or recombinant DNA which encodes a biologically active proteinor polypeptide and which is capable of hybridizing under appropriateconditions with the DNA sequences described herein. Said biologicallyactive protein or polypeptide can be an intact antigen, or fragment, andhave an amino acid sequence disclosed in, e.g., SEQ ID NO: 2, 4, 6, or8, or any of the foregoing in association with SEQ ID NO: 10,particularly a mature, secreted polypeptide. Further, this inventioncovers the use of isolated or recombinant DNA, or fragments thereof,which encode proteins which exhibit high identity to a secreted IL-D80or IL-27. The isolated DNA can have the respective regulatory sequencesin the 5′ and 3′ flanks, e.g., promoters, enhancers, poly-A additionsignals, and others. Alternatively, expression may be effected byoperably linking a coding segment to a heterologous promoter, e.g., byinserting a promoter upstream from an endogenous gene.

An “isolated” nucleic acid is a nucleic acid, e.g., an RNA, DNA, or amixed polymer, which is substantially separated from other componentswhich naturally accompany a native sequence, e.g., ribosomes,polymerases, and/or flanking genomic sequences from the originatingspecies. The term embraces a nucleic acid sequence which has beenremoved from its naturally occurring environment, and includesrecombinant or cloned DNA isolates and chemically synthesized analogs oranalogs biologically synthesized by heterologous systems. Asubstantially pure molecule includes isolated forms of the molecule.Generally, the nucleic acid will be in a vector or fragment less thanabout 50 kb, usually less than about 30 kb, typically less than about 10kb, and preferably less than about 6 kb.

An isolated nucleic acid will generally be a homogeneous composition ofmolecules, but will, in some embodiments, contain minor heterogeneity.This heterogeneity is typically found at the polymer ends or portionsnot critical to a desired biological function or activity.

A “recombinant” nucleic acid is defined either by its method ofproduction or its structure. In reference to its method of production,e.g., a product made by a process, the process is use of recombinantnucleic acid techniques, e.g., involving human intervention in thenucleotide sequence, typically selection or production. Alternatively,it can be a nucleic acid made by generating a sequence comprising fusionof two fragments which are not naturally contiguous to each other, butis meant to exclude products of nature, e.g., naturally occurringmutants. Thus, e.g., products made by transforming cells with anyunnaturally occurring vector is encompassed, as are nucleic acidscomprising sequence derived using any synthetic oligonucleotide process.Such is often done to replace a codon with a redundant codon encodingthe same or a conservative amino acid, while typically introducing orremoving a sequence recognition site.

Alternatively, it is performed to join together nucleic acid segments ofdesired functions to generate a single genetic entity comprising adesired combination of functions not found in the commonly availablenatural forms. Restriction enzyme recognition sites are often the targetof such artificial manipulations, but other site specific targets, e.g.,promoters, DNA replication sites, regulation sequences, controlsequences, or other useful features may be incorporated by design. Asimilar concept is intended for a recombinant, e.g., fusion,polypeptide. Specifically included are synthetic nucleic acids which, bygenetic code redundancy, encode polypeptides similar to fragments ofthese antigens, and fusions of sequences from various different speciesor polymorphic variants.

A significant “fragment” in a nucleic acid context is a contiguoussegment of at least about 17 nucleotides, generally at least about 22nucleotides, ordinarily at least about 29 nucleotides, more often atleast about 35 nucleotides, typically at least about 41 nucleotides,usually at least about 47 nucleotides, preferably at least about 55nucleotides, and in particularly preferred embodiments will be at leastabout 60 or more nucleotides, e.g., 67, 73, 81, 89, 95, 150, 200, 250,300, 500, etc.

A DNA which codes for an IL-D80 or IL-27 protein will be particularlyuseful to identify genes, mRNA, and cDNA species which code for relatedor similar proteins, as well as DNAs which code for homologous proteinsfrom different species. There will be homologs in other species,including primates, rodents, canines, felines, birds, and fish. VariousIL-D80 or IL-27 proteins should be homologous and are encompassedherein. However, even proteins that have a more distant evolutionaryrelationship to the antigen can readily be isolated under appropriateconditions using these sequences if they are sufficiently homologous.Primate IL-D80 or IL-27 proteins are of particular interest.

Recombinant clones derived from the genomic sequences, e.g., containingintrons, will be useful for transgenic studies, including, e.g.,transgenic cells and organisms, and for gene therapy. See, e.g., Goodnow(1992) “Transgenic Animals” in Roitt (ed.) Encyclopedia of Immunology,Academic Press, San Diego, pp. 1502-1504; Travis (1992) Science256:1392-1394; Kuhn, et al. (1991) Science 254:707-710; Capecchi (1989)Science 244:1288; Robertson (ed. 1987) Teratocarcinomas and EmbryonicStem Cells: A Practical Approach, IRL Press, Oxford; Rosenberg (1992) J.Clinical Oncology 10:180-199; and Cournoyer and Caskey (1993) Ann. Rev.Immunol. 11:297-329. Alternatively, expression may be effected byoperably linking a coding segment to a heterologous promoter, e.g., byinserting a promoter upstream from an endogenous gene. See, e.g., Treco,et al. WO96/29411 or U.S. Ser. No. 08/406,030.

Substantial homology, e.g., identity, in the nucleic acid sequencecomparison context means either that the segments, or theircomplementary strands, when compared, are identical when optimallyaligned, with appropriate nucleotide insertions or deletions, in atleast about 50% of the nucleotides, generally at least about 58%,ordinarily at least about 65%, often at least about 71%, typically atleast about 77%, usually at least about 85%, preferably at least about95 to 98% or more, and in particular embodiments, as high as about 99%or more of the nucleotides. Alternatively, substantial homology existswhen the segments will hybridize under selective hybridizationconditions, to a strand, or its complement, typically using a sequenceof IL-D80 or IL-27, e.g., in SEQ ID NO: 1, 3, 5, or 7, or any of theforegoing in association with SEQ ID NO: 9. Typically, selectivehybridization will occur when there is at least about 55% identity overa stretch of at least about 30 nucleotides, preferably at least about75% over a stretch of about 25 nucleotides, and most preferably at leastabout 90% over about 20 nucleotides. See, Kanehisa (1984) Nuc. AcidsRes. 12:203-213. The length of identity comparison, as described, may beover longer stretches, and in certain embodiments will be over a stretchof at least about 17 nucleotides, usually at least about 28 nucleotides,typically at least about 40 nucleotides, and preferably at least about75 to 100 or more nucleotides.

Stringent conditions, in referring to homology in the hybridizationcontext, will be stringent combined conditions of salt, temperature,organic solvents, and other parameters, typically those controlled inhybridization reactions. Stringent temperature conditions will usuallyinclude temperatures in excess of about 30° C., usually in excess ofabout 37° C., typically in excess of about 55° C., 60° C., or 65° C.,and preferably excess of about 70° C. Stringent salt conditions willordinarily be less than about 1000 mM, usually less than about 400 mM,typically less than about 250 mM, preferably less than about 150 mM,including about 100, 50, or even 20 mM. However, the combination ofparameters is much more important than the measure of any singleparameter. See, e.g., Wetmur and Davidson (1968) J. Mol. Biol.31:349-370. Hybridization under stringent conditions should give abackground of at least 2-fold over background, preferably at least 3-5or more.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optical alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith and Waterman (1981) Adv. Appl.Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch(1970) J. Mol. Biol. 48:443, by the search for similarity method ofPearson and Lipman (1988) Proc. Nat'l Acad. Sci. USA 85:2444, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by visual inspection (seegenerally Ausubel et al., supra).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendrogram showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng and Doolittle (1987) J. Mol. Evol.35:351-360. The method used is similar to the method described byHiggins and Sharp (1989) CABIOS 5:151-153. The program can align up to300 sequences, each of a maximum length of 5,000 nucleotides or aminoacids. The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster is then aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences arealigned by a simple extension of the pairwise alignment of twoindividual sequences. The final alignment is achieved by a series ofprogressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. For example, a reference sequence can be compared to othertest sequences to determine the percent sequence identity relationshipusing the following parameters: default gap weight (3.00), default gaplength weight (0.10), and weighted end gaps.

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described Altschul, et al. (1990) J. Mol. Biol. 215:403-410. Softwarefor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information (http:www.ncbi.nlm.nih.gov/). Thisalgorithm involves first identifying high scoring sequence pairs (HSPs)by identifying short words of length W in the query sequence, whicheither match or satisfy some positive-valued threshold score T whenaligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul, et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are thenextended in both directions along each sequence for as far as thecumulative alignment score can be increased. Extension of the word hitsin each direction are halted when: the cumulative alignment score fallsoff by the quantity X from its maximum achieved value; the cumulativescore goes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLAST program uses asdefaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Nat'l Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparisonof both strands.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin and Altschul (1993) Proc. Nat'l Acad.Sci. USA 90:5873-5787). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

A further indication that two nucleic acid sequences of polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid, as described below. Thus, apolypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions, as described below.

IL-D80 or IL-27 from other mammalian species can be cloned and isolatedby cross-species hybridization of closely related species. Homology maybe relatively low between distantly related species, and thushybridization of relatively closely related species is advisable.Alternatively, preparation of an antibody preparation which exhibitsless species specificity may be useful in expression cloning approaches.

VII. Making IL-D80 or IL-27; Mimetics

DNA which encodes the IL-D80 or IL-27 or fragments thereof can beobtained by chemical synthesis, screening cDNA libraries, or screeninggenomic libraries prepared from a wide variety of cell lines or tissuesamples. See, e.g., Okayama and Berg (1982) Mol. Cell. Biol. 2:161-170;Gubler and Hoffman (1983) Gene 25:263-269; and Glover (ed. 1984) DNACloning: A Practical Approach, IRL Press, Oxford. Alternatively, thesequences provided herein provide useful PCR primers or allow syntheticor other preparation of suitable genes encoding an IL-D80 or IL-27;including naturally occurring embodiments.

This DNA can be expressed in a wide variety of host cells for thesynthesis of a full-length IL-D80 or IL-27 or fragments which can inturn, e.g., be used to generate polyclonal or monoclonal antibodies; forbinding studies; for construction and expression of modified molecules;and for structure/function studies. There may be a need for a chaparoneprotein for efficient secretion, or additional steps may be necessary toretrieve the protein from the intracellular compartment.

Vectors, as used herein, comprise plasmids, viruses, bacteriophage,integratable DNA fragments, and other vehicles which enable theintegration of DNA fragments into the genome of the host. See, e.g.,Pouwels, et al. (1985 and Supplements) Cloning Vectors: A LaboratoryManual, Elsevier, N.Y.; and Rodriguez, et al. (eds. 1988) Vectors: ASurvey of Molecular Cloning Vectors and Their Uses, Buttersworth,Boston, Mass.

For purposes of this invention, DNA sequences are operably linked whenthey are functionally related to each other. For example, DNA for apresequence or secretory leader is operably linked to a polypeptide ifit is expressed as a preprotein or participates in directing thepolypeptide to the cell membrane or in secretion of the polypeptide. Apromoter is operably linked to a coding sequence if it controls thetranscription of the polypeptide; a ribosome binding site is operablylinked to a coding sequence if it is positioned to permit translation.Usually, operably linked means contiguous and in reading frame, however,certain genetic elements such as repressor genes are not contiguouslylinked but still bind to operator sequences that in turn controlexpression. See, e.g., Rodriguez, et al., Chapter 10, pp. 205-236;Balbas and Bolivar (1990) Methods in Enzymology 185:14-37; and Ausubel,et al. (1993) Current Protocols in Molecular Biology, Greene and Wiley,N.Y.

Representative examples of suitable expression vectors include pCDNA1;pCD, see Okayama, et al. (1985) Mol. Cell Biol. 5:1136-1142; pMC1neoPoly-A, see Thomas, et al. (1987) Cell 51:503-512; and a baculovirusvector such as pAC 373 or pAC 610. See, e.g., Miller (1988) Ann. Rev.Microbiol. 42:177-199.

It will often be desired to express an IL-D80 or IL-27 polypeptide in asystem which provides a specific or defined glycosylation pattern. See,e.g., Luckow and Summers (1988) Bio/Technology 6:47-55; and Kaufman(1990) Meth. Enzymol. 185:487-511.

The IL-D80 or IL-27, or a fragment thereof, may be engineered to bephosphatidyl inositol (PI) linked to a cell membrane, but can be removedfrom membranes by treatment with a phosphatidyl inositol cleavingenzyme, e.g., phosphatidyl inositol phospholipase-C. This releases theantigen in a biologically active form, and allows purification bystandard procedures of protein chemistry. See, e.g., Low (1989) Biochim.Biophys. Acta 988:427-454; Tse, et al. (1985) Science 230:1003-1008; andBrunner, et al. (1991) J. Cell Biol. 114:1275-1283.

Now that the IL-D80 or IL-27 has been characterized, fragments orderivatives thereof can be prepared by conventional processes forsynthesizing peptides. These include processes such as are described inStewart and Young (1984) Solid Phase Peptide Synthesis, Pierce ChemicalCo., Rockford, Ill.; Bodanszky and Bodanszky (1984) The Practice ofPeptide Synthesis, Springer-Verlag, New York; Bodanszky (1984) ThePrinciples of Peptide Synthesis, Springer-Verlag, New York; andVillafranca (ed. 1991) Techniques in Protein Chemistry II, AcademicPress, San Diego, Calif.

VIII. Uses

The present invention provides reagents which will find use indiagnostic applications as described elsewhere herein, e.g., in IL-D80or IL-27 mediated conditions, or below in the description of kits fordiagnosis. The gene may be useful in forensic sciences, e.g., todistinguish rodent from human, or as a marker to distinguish betweendifferent cells exhibiting differential expression or modificationpatterns.

This invention also provides reagents with significant commercial and/ortherapeutic potential. The IL-D80 or IL-27 (naturally occurring orrecombinant), fragments thereof, and antibodies thereto, along withcompounds identified as having binding affmity to IL-D80 or IL-27,should be useful as reagents for teaching techniques of molecularbiology, immunology, or physiology. Appropriate kits may be preparedwith the reagents, e.g., in practical laboratory exercises in productionor use of proteins, antibodies, cloning methods, histology, etc.

The reagents will also be useful in the treatment of conditionsassociated with abnormal physiology or development, includinginflammatory conditions. They may be useful in vitro tests for presenceor absence of interacting components, which may correlate with successof particular treatment strategies. In particular, modulation ofphysiology of various, e.g., hematopoietic or lymphoid, cells will beachieved by appropriate methods for treatment using the compositionsprovided herein. See, e.g., Thomson (ed. 1998) The Cytokine Handbook (3ded.) Academic Press, San Diego; Metcalf and Nicola (1995) TheHematopoietic Colony Stimulating Factors Cambridge University Press; andAggarwal and Gutterman (1991) Human Cytokines Blackwell Pub.

For example, a disease or disorder associated with abnormal expressionor abnormal signaling by an IL-D80 or IL-27 should be a likely targetfor an agonist or antagonist. Similarly, the binding partner of theIL-27 composite cytokine, WSX-1/TCCR, should also be a target. The newcytokine should play a role in regulation or development ofhematopoietic cells, e.g., lymphoid cells, which affect immunologicalresponses, e.g., inflammation and/or autoimmune disorders.Alternatively, it may affect vascular physiology or development, orneuronal effects.

In particular, the cytokine should mediate, in various contexts,cytokine synthesis by the cells, proliferation, etc. Antagonists ofIL-D80 or IL-27, such as mutein variants of a naturally occurring formof IL-D80 or IL-27 or blocking antibodies, may provide a selective andpowerful way to block immune responses, e.g., in situations asinflammatory or autoimmune responses. See also Samter, et al. (eds.)Immunological Diseases vols. 1 and 2, Little, Brown and Co.

Various abnormal conditions are known in different cell types which willproduce IL-D80 or IL-27, e.g., as evaluated by mRNA expression byNorthern blot analysis. See Berkow (ed.) The Merck Manual of Diagnosisand Therapy, Merck & Co., Rahway, N.J.; Thorn, et al. Harrison'sPrinciples of Internal Medicine, McGraw-Hill, N.Y.; and Weatherall, etal. (eds.) Oxford Textbook of Medicine, Oxford University Press, Oxford.Many other medical conditions and diseases involve activation bymacrophages or monocytes, and many of these will be responsive totreatment by an agonist or antagonist provided herein. See, e.g., Stitesand Terr (eds.; 1991) Basic and Clinical Immunology Appleton and Lange,Norwalk, Conn.; and Samter, et al. (eds.) Immunological Diseases Little,Brown and Co. These problems should be susceptible to prevention ortreatment using compositions provided herein.

IL-D80 or IL-27, antagonists, antibodies, etc., can be purified and thenadministered to a patient, veterinary or human. These reagents can becombined for therapeutic use with additional active or inertingredients, e.g., in conventional pharmaceutically acceptable carriersor diluents, e.g., immunogenic adjuvants, along with physiologicallyinnocuous stabilizers, excipients, or preservatives. These combinationscan be sterile filtered and placed into dosage forms as bylyophilization in dosage vials or storage in stabilized aqueouspreparations. This invention also contemplates use of antibodies orbinding fragments thereof, including forms which are not complementbinding.

Drug screening using IL-D80, IL-27, WSX-1/TCCR or fragments thereof canbe performed to identify compounds having binding affinity to or otherrelevant biological effects on IL-D80 or IL-27 functions, includingisolation of associated components. Subsequent biological assays canthen be utilized to determine if the compound has intrinsic stimulatingactivity and is therefore a blocker or antagonist in that it blocks theactivity of the cytokine. Likewise, a compound having intrinsicstimulating activity can activate the signal pathway and is thus anagonist in that it simulates the activity of IL-D80 or IL-27. Thisinvention further contemplates the therapeutic use of blockingantibodies to IL-D80, IL-27, or WSX-1/TCCR as antagonists and ofstimulatory antibodies as agonists. This approach should be particularlyuseful with other IL-D80 or IL-27 species variants.

The quantities of reagents necessary for effective therapy will dependupon many different factors, including means of administration, targetsite, physiological state of the patient, and other medicantsadministered. Thus, treatment dosages should be titrated to optimizesafety and efficacy. Typically, dosages used in vitro may provide usefulguidance in the amounts useful for in situ administration of thesereagents. Animal testing of effective doses for treatment of particulardisorders will provide further predictive indication of human dosage.Various considerations are described, e.g., in Gilman, et al. (eds.)Goodman and Gilman's: The Pharmacological Bases of Therapeutics, latestEd., Pergamon Press; and Remington's Pharmaceutical Sciences, latested., Mack Publishing Co., Easton, Pa. Methods for administration arediscussed therein and below, e.g., for oral, intravenous,intraperitoneal, or intramuscular administration, transdermal diffusion,and others. Pharmaceutically acceptable carriers will include water,saline, buffers, and other compounds described, e.g., in the MerckIndex, Merck & Co., Rahway, N.J. Dosage ranges would ordinarily beexpected to be in amounts lower than 1 mM concentrations, typically lessthan about 10 μM concentrations, usually less than about 100 nM,preferably less than about 10 pM (picomolar), and most preferably lessthan about 1 fM (femtomolar), with an appropriate carrier. Slow releaseformulations, or a slow release apparatus will often be utilized forcontinuous or long term administration. See, e.g., Langer (1990) Science249:1527-1533.

IL-D80 or IL-27, fragments thereof, and antibodies to it or itsfragments, antagonists, and agonists, may be administered directly tothe host to be treated or, depending on the size of the compounds, itmay be desirable to conjugate them to carrier proteins such as ovalbuminor serum albumin prior to their administration. Therapeutic.formulations may be administered in many conventional dosageformulations. While it is possible for the active ingredient to beadministered alone, it is preferable to present it as a pharmaceuticalformulation. Formulations typically comprise at least one activeingredient, as defined above, together with one or more acceptablecarriers thereof. Each carrier should be both pharmaceutically andphysiologically acceptable in the sense of being compatible with theother ingredients and not injurious to the patient. Formulations includethose suitable for oral, rectal, nasal, topical, or parenteral(including subcutaneous, intramuscular, intravenous and intradermal)administration. The formulations may conveniently be presented in unitdosage form and may be prepared by any methods well known in the art ofpharmacy. See, e.g., Gilman, et al. (eds. 1990) Goodman and Gilman's:The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; andRemington's Pharmaceutical Sciences, 17th ed. (1990), Mack PublishingCo., Easton, Pa.; Avis, et al. (eds. 1993) Pharmaceutical Dosage Forms:Parenteral Medications, Dekker, N.Y.; Lieberman, et al. (eds. 1990)Pharmaceutical Dosage Forms: Tablets, Dekker, N.Y.; and Lieberman, etal. (eds. 1990) Pharmaceutical Dosage Forms: Disperse Systems, Dekker,N.Y. The therapy of this invention may be combined with or used inassociation with other agents, e.g., other cytokines, including IL-12,or its antagonists.

Both naturally occurring and recombinant forms of the IL-D80 or IL-27sof this invention are particularly useful in kits and assay methodswhich are capable of screening compounds for binding activity to theproteins. Several methods of automating assays have been developed inrecent years so as to permit screening of tens of thousands of compoundsin a short period. See, e.g., Fodor, et al. (1991) Science 251:767-773,which describes means for testing of binding affinity by a plurality ofdefined polymers synthesized on a solid substrate. The development ofsuitable assays can be greatly facilitated by the availability of largeamounts of purified, soluble IL-D80 or IL-27 as provided by thisinvention.

Other methods can be used to determine the critical residues in IL-D80or IL-27 receptor interactions. Mutational analysis can be performed,e.g., see Somoza, et al. (1993) J. Exptl. Med. 178:549-558, to determinespecific residues critical in the interaction and/or signaling. PHD(Rost and Sander (1994) Proteins 19:55-72) and DSC (King and Sternberg(1996) Protein Sci. 5:2298-2310) can provide secondary structurepredictions of α-helix (H), β-strand (E), or coil (L). Helices A and Dare most important in receptor interaction, with the D helix the moreimportant region. Boundaries for the various helices are indicatedabove. Surface exposed residues would affect receptor binding, whileembedded residues would affect general structure.

For example, antagonists can normally be found once the antigen has beenstructurally defined, e.g., by tertiary structure data. Testing ofpotential interacting analogs is now possible upon the development ofhighly automated assay methods using a purified IL-D80 or IL-27. Inparticular, new agonists and antagonists will be discovered by usingscreening techniques described herein. Of particular importance arecompounds found to have a combined binding affinity for a spectrum ofIL-D80 or IL-27 molecules, e.g., compounds which can serve asantagonists for species variants of IL-D80 or IL-27.

One method of drug screening utilizes eukaryotic or prokaryotic hostcells which are stably transformed with recombinant DNA moleculesexpressing an IL-D80 or IL-27. Cells may be isolated which express anIL-D80 or IL-27 in isolation from other molecules. Such cells, either inviable or fixed form, can be used for standard binding partner bindingassays. See also, Parce, et al. (1989) Science 246:243-247; and Owicki,et al. (1990) Proc. Nat'l Acad. Sci. USA 87:4007-4011, which describesensitive methods to detect cellular responses.

Another technique for drug screening involves an approach which provideshigh throughput screening for compounds having suitable binding affinityto an IL-D80 or IL-27 and is described in detail in Geysen, EuropeanPatent Application 84/03564, published on Sep. 13, 1984. First, largenumbers of different small peptide test compounds are synthesized on asolid substrate, e.g., plastic pins or some other appropriate surface,see Fodor, et al. (1991). Then all the pins are reacted withsolubilized, unpurified or solubilized, purified IL-D80 or IL-27, andwashed. The next step involves detecting bound IL-D80 or IL-27.

Rational drug design may also be based upon structural studies of themolecular shapes of the IL-D80 or IL-27 and other effectors or analogs.Effectors may be other proteins which mediate other functions inresponse to binding, or other proteins which normally interact withIL-D80 or IL-27, e.g., a receptor. One means for determining which sitesinteract with specific other proteins is a physical structuredetermination, e.g., x-ray crystallography or 2 dimensional NMRtechniques. These will provide guidance as to which amino acid residuesform molecular contact regions, as modeled, e.g., against othercytokine-receptor models. For a detailed description of proteinstructural determination, see, e.g., Blundell and Johnson (1976) ProteinCrystallography, Academic Press, New York.

IX. Kits

This invention also contemplates use of IL-D80 or IL-27 proteins,fragments thereof, peptides, and their fusion products in a variety ofdiagnostic kits and methods for detecting the presence of another IL-D80or IL-27 or binding partner. Typically the kit will have a compartmentcontaining either a defined IL-D80 or IL-27 peptide or gene segment or areagent which recognizes one or the other, e.g., IL-D80 or IL-27fragments or antibodies.

A kit for determining the binding affinity of a test compound to anIL-D80 or IL-27 would typically comprise a test compound; a labeledcompound, for example a binding partner or antibody having known bindingaffinity for IL-D80 or IL-27; a source of IL-D80 or IL-27 (naturallyoccurring or recombinant); and a means for separating bound from freelabeled compound, such as a solid phase for immobilizing the molecule.Once compounds are screened, those having suitable binding affinity tothe antigen can be evaluated in suitable biological assays, as are wellknown in the art, to determine whether they act as agonists orantagonists to the IL-D80 or IL-27 signaling pathway. The availabilityof recombinant IL-D80 or IL-27 polypeptides also provide well definedstandards for calibrating such assays.

A preferred kit for determining the concentration of, e.g., an IL-D80 orIL-27 in a sample would typically comprise a labeled compound, e.g.,binding partner or antibody, having known binding affinity for theantigen, a source of cytokine (naturally occurring or recombinant) and ameans for separating the bound from free labeled compound, e.g., a solidphase for immobilizing the IL-D80 or IL-27. Compartments containingreagents, and instructions, will normally be provided.

Antibodies, including antigen binding fragments, specific for the IL-D80or IL-27 or fragments are useful in diagnostic applications to detectthe presence of elevated levels of IL-D80 or IL-27 and/or its fragments.Such diagnostic assays can employ lysates, live cells, fixed cells,immunofluorescence, cell cultures, body fluids, and further can involvethe detection of antigens related to the antigen in serum, or the like.Diagnostic assays may be homogeneous (without a separation step betweenfree reagent and antigen-binding partner complex) or heterogeneous (witha separation step). Various commercial assays exist, such asradioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA),enzyme immunoassay (EIA), enzyme-multiplied immunoassay technique(EMIT), substrate-labeled fluorescent immunoassay (SLFIA), and the like.See, e.g., Van Vunakis, et al. (1980) Meth Enzymol. 70:1-525; Harlow andLane (1980) Antibodies: A Laboratory Manual, CSH Press, N.Y; andColigan, et al. (eds. 1993) Current Protocols in Immunology, Greene andWiley, N.Y.

Anti-idiotypic antibodies may have similar use to diagnose presence ofantibodies against an IL-D80 or IL-27, as such may be diagnostic ofvarious abnormal states. For example, overproduction of IL-D80 or IL-27may result in production of various immunological reactions which may bediagnostic of abnormal physiological states, particularly inproliferative cell conditions such as cancer or abnormal activation ordifferentiation. Moreover, the distribution pattern available providesinformation that the cytokine is expressed in pancreatic islets,suggesting the possibility that the cytokine may be involved in functionof that organ, e.g., in a diabetes relevant medical condition.

Frequently, the reagents for diagnostic assays are supplied in kits, soas to optimize the sensitivity of the assay. For the subject invention,depending upon the nature of the assay, the protocol, and the label,either labeled or unlabeled antibody or binding partner, or labeledIL-D80 or IL-27 is provided. This is usually in conjunction with otheradditives, such as buffers, stabilizers, materials necessary for signalproduction such as substrates for enzymes, and the like. Preferably, thekit will also contain instructions for proper use and disposal of thecontents after use. Typically the kit has compartments for each usefulreagent. Desirably, the reagents are provided as a dry lyophilizedpowder, where the reagents may be reconstituted in an aqueous mediumproviding appropriate concentrations of reagents for performing theassay.

Many of the aforementioned constituents of the drug screening and thediagnostic assays may be used without modification or may be modified ina variety of ways. For example, labeling may be achieved by covalentlyor non-covalently joining a moiety which directly or indirectly providesa detectable signal. In any of these assays, the binding partner, testcompound, IL-D80 or IL-27, or antibodies thereto can be labeled eitherdirectly or indirectly. Possibilities for direct labeling include labelgroups: radiolabels such as ¹²⁵I, enzymes (U.S. Pat. No. 3,645,090) suchas peroxidase and alkaline phosphatase, and fluorescent labels (U.S.Pat. No. 3,940,475) capable of monitoring the change in fluorescenceintensity, wavelength shift, or fluorescence polarization. Possibilitiesfor indirect labeling include biotinylation of one constituent followedby binding to avidin coupled to one of the above label groups.

There are also numerous methods of separating the bound from the freeIL-D80 or IL-27, or alternatively the bound from the free test compound.The IL-D80 or IL-27 can be immobilized on various matrixes followed bywashing. Suitable matrixes include plastic such as an ELISA plate,filters, and beads. See, e.g., Coligan, et al. (eds. 1993) CurrentProtocols in Immunology, Vol. 1, Chapter 2, Greene and Wiley, NY. Othersuitable separation techniques include, without limitation, thefluorescein antibody magnetizable particle method described in Rattle,et al. (1984) Clin. Chem. 30:1457-1461, and the double antibody magneticparticle separation as described in U.S. Pat. No. 4,659,678.

Methods for linking proteins or their fragments to the various labelshave been extensively reported in the literature and do not requiredetailed discussion here. Many of the techniques involve the use ofactivated carboxyl groups either through the use of carbodiimide oractive esters to form peptide bonds, the formation of thioethers byreaction of a mercapto group with an activated halogen such aschloroacetyl, or an activated olefin such as maleimide, for linkage, orthe like. Fusion proteins will also find use in these applications.

Another diagnostic aspect of this invention involves use ofoligonucleotide or polynucleotide sequences taken from the sequence ofan IL-D80 or IL-27. These sequences can be used as probes for detectinglevels of the IL-D80 or IL-27 message in samples from patients suspectedof having an abnormal condition, e.g., inflammatory or autoimmune. Sincethe cytokine may be a marker or mediator for activation, it may beuseful to determine the numbers of activated cells to determine, e.g.,when additional therapy may be called for, e.g., in a preventativefashion before the effects become and progress to significance. Thepreparation of both RNA and DNA nucleotide sequences, the labeling ofthe sequences, and the preferred size of the sequences has receivedample description and discussion in the literature. See, e.g.,Langer-Safer, et al. (1982) Proc. Nat'l. Acad. Sci. 79:4381-4385; Caskey(1987) Science 236:962-967; and Wilchek et al. (1988) Anal. Biochem.171:1-32.

Diagnostic kits which also test for the qualitative or quantitativeexpression of other molecules are also contemplated. Diagnosis orprognosis may depend on the combination of multiple indications used asmarkers. Thus, kits may test for combinations of markers. See, e.g.,Viallet, et al. (1989) Progress in Growth Factor Res. 1:89-97. Otherkits may be used to evaluate other cell subsets.

X. Isolating an IL-D80 or IL-27 Receptor

Having isolated a ligand of a specific ligand-receptor interaction,methods exist for isolating the receptor. See, Gearing, et al. (1989)EMBO J. 8:3667-3676. For example, means to label the IL-D80 or IL-27cytokine without interfering with the binding to its receptor can bedetermined. For example, an affinity label can be fused to either theamino- or carboxyl-terminus of the ligand. Such label may be a FLAGepitope tag, or, e.g., an Ig or Fc domain. An expression library can bescreened for specific binding of the cytokine, e.g., by cell sorting, orother screening to detect subpopulations which express such a bindingcomponent. See, e.g., Ho, et al. (1993) Proc. Nat'l Acad. Sci. USA90:11267-11271; and Liu, et al. (1994) J. Immunol. 152:1821-29.Alternatively, a panning method may be used. See, e.g., Seed and Aruffo(1987) Proc. Nat'l Acad. Sci. USA 84:3365-3369.

Protein cross-linking techniques with label can be applied to isolatebinding partners of the IL-D80 or IL-27 cytokine. This would allowidentification of proteins which specifically interact with thecytokine, e.g., in a ligand-receptor. like manner. It has been shown, asnoted below, that the IL-27 composite cytokine binds at least to anIL-12R-like subunit known as WSX-1/TCCR.

FACS analysis of detectably stained IL-D80, EBI3, and WSX-1/TCCRmolecules led to the finding that these molecules are components in areceptor subunit/ligand complex. Specifically, the composite cytokine ofE-tagged hIL-D80 (hIL-D80E) and F-tagged (FLAG-tagged) hEBI3 (FhEBI3)binds to Baf3 cells expressing an F-tagged version of WSX-1/TCCR, alsoreferred to as hNR30. The cells were stained using anti-E mAb and aPE-conjugated anti-mouse Fab₂ fragment. Co-immunoprecipitationexperiments also indicated that hIL-27 could be immunoprecipitated withR-tagged (RGSH₆-tagged) soluble WSX-1/TCCR (shNR30R). Alternatively,shNR30R could be co-immunoprecipated in the presence of hIL-D80E/FhEBI3complex using anti-E or anti-F mAbs. These experiments establish thatWSX-1/TCCR is a receptor component of the IL-27 composite cytokine.Recent evidence shows that disrupting the WSX-1/TCCR gene in miceresults in lowered expression of IFNγ, which is a critical cytokine inthe mediation of pro-inflammatory functions. These mice were unable tomount a Th1 response (See, e.g., Chen, et al. (2000) Nature407:916-920).

Experimental data indicates a possible role for the IL-27 compositecytokine in driving an inflammatory response. The expression profile ofEBI3 and IL-D80 overlaps in monocytes, macrophages, and dendritic cells,indicating that the composite cytokine is primarily produced by antigentpresenting cells (APCs) of the immune system. EBI3 has been shown to beupregulated in colonic tissue of patients suffering from gutinflammation disorders, e.g., ulcerative colitis, suggesting that thecomposite cytokine may also be involved.

Taken together the above indicates a role for the composite cytokine andits associated receptor subunit WSX-1/TCCR in inflammatory responses.Therefore antagonizing the function of any of the components in thereceptor subunit:ligand complex should have a beneficial effect ininflammatory diseases, e.g., inflammatory bowel disease, rheumatoidarthritis, etc.

EXAMPLES

I. General Methods

Many of the standard methods below are described or referenced, e.g., inManiatis, et al. (1982) Molecular Cloning, A Laboratory Manual ColdSpring Harbor Laboratory, Cold Spring Harbor Press, NY; Sambrook, et al.(1989) Molecular Cloning: A Laboratory Manual (2d ed.) Vols. 1-3, CSHPress, NY; Ausubel, et al., Biology Greene Publishing Associates,Brooklyn, N.Y.; or Ausubel, et al. (1987 and Supplements) CurrentProtocols in Molecular Biology Wiley/Greene, NY; Innis, et al. (eds.1990) PCR Protocols: A Guide to Methods and Applications Academic Press,NY. Methods for protein purification include such methods as ammoniumsulfate precipitation, column chromatography, electrophoresis,centrifugation, crystallization, and others. See, e.g., Ausubel, et al.(1987 and periodic supplements); Deutscher (1990) “Guide to ProteinPurification,” Methods in Enzymology vol. 182, and other volumes in thisseries; Coligan, et al. (1995 and supplements) Current Protocols inProtein Science John Wiley and Sons, New York, N.Y.; P. Matsudaira (ed.1993) A Practical Guide to Protein and Peptide Purification forMicrosequencing, Academic Press, San Diego, Calif.; and manufacturer'sliterature on use of protein purification products, e.g., Pharmacia,Piscataway, N.J., or Bio-Rad, Richmond, Calif. Combination withrecombinant techniques allow fusion to appropriate segments (epitopetags), e.g., to a FLAG sequence or an equivalent which can be fused,e.g., via a protease-removable sequence. See, e.g., Hochuli (1989)Chemische Industrie 12:69-70; Hochuli (1990) “Purification ofRecombinant Proteins with Metal Chelate Absorbent” in Setlow (ed.)Genetic Engineering, Principle and Methods 12:87-98, Plenum Press, NY;and Crowe, et al. (1992) QIAexpress: The High Level Expression & ProteinPurification System QUIAGEN, Inc., Chatsworth, Calif.

Standard immunological techniques are described, e.g., in Hertzenberg,et al. (eds. 1996) Weir's Handbook of Experimental Immunology vols. 1-4,Blackwell Science;

Coligan (1991) Current Protocols in Immunology Wiley/Greene, NY; andMethods in Enzymology vols. 70, 73, 74, 84, 92, 93, 108, 116, 121, 132,150, 162, and 163. Cytokine assays are described, e.g., in Thomson (ed.1998) The Cytokine Handbook (3d ed.) Academic Press, San Diego;Mire-Sluis and Thorpe (1998) Cytokines Academic Press, San Diego;Metcalf and Nicola (1995) The Hematopoietic Colony Stimulating FactorsCambridge University Press; and Aggarwal and Gutterman (1991) HumanCytokines Blackwell Pub.

Assays for vascular biological activities are well known in the art.They will cover angiogenic and angiostatic activities in tumor, or othertissues, e.g., arterial smooth muscle proliferation (see, e.g., Koyoma,et al. (1996) Cell 87:1069-1078), monocyte adhesion to vascularepithelium (see McEvoy, et al. (1997) J. Exp. Med. 185:2069-2077), etc.See also Ross (1993) Nature 362:801-809; Rekhter and Gordon (1995) Am.J. Pathol. 147:668-677; Thyberg, et al. (1990) Atherosclerosis10:966-990; and Gumbiner (1996) Cell 84:345-357.

Assays for neural cell biological activities are described, e.g., inWouterlood (ed. 1995) Neuroscience Protocols modules 10, Elsevier;Methods in Neurosciences Academic Press; and Neuromethods Humana Press,Totowa, N.J. Methodology of developmental systems is described, e.g., inMeisami (ed.) Handbook of Human Growth and Developmental Biology CRCPress; and Chrispeels (ed.) Molecular Techniques and Approaches inDevelopmental Biology Interscience.

FACS analyses are described in Melarned, et al. (1990) Flow Cytometryand Sorting Wiley-Liss, Inc., New York, N.Y.; Shapiro (1988) PracticalFlow Cytometry Liss, New York, N.Y.; and Robinson, et al. (I1993)Handbook of Flow Cytometry Methods Wiley-Liss, New York, N.Y.

II. Cloning of Human IL-D80

The sequences of primate, e.g., human, genes are provided in SEQ ID NO:1, 3, 5, and 7. These sequences are derived from a sequence database.These sequences allow preparation of PCR primers, or probes, todetermine cellular distribution of the gene. These sequences allowisolation of genomic DNA which encode the message.

Using the probe or PCR primers, various tissues or cell types are probedto determine cellular distribution. PCR products are cloned using, e.g.,a TA cloning kit (Invitrogen). The resulting cDNA plasmids- aresequenced from both termini on an automated sequencer (AppliedBiosystems).

A structural alignment of available IL-6 family cytokine folds (CNTF,LIF, IL-6, OSM and GCSF) from FSSP (see, e.g., Holm and Sander (1998)Nucleic Acids Res. 26:316-319 was profile-aligned to other sequences(including distant species variants of the aforementioned cytokines,plus CT-1, GPA and viral IL-6's) with Clustal X (see, e.g., Thompson, etal. (1997) Nucleic Acids Res. 25:4876-4882) with some manual adjustment.A weighted profile (see, e.g., Thompson, et al. (1994) Nucleic AcidsRes. 22:4673-4680) of the most conserved region of the fold, theC-terminal D-helix segment, a ˜40 amino acid block, was created. Fastscans of sequence databases on a Bioccelerator machine (Compugen, TelAviv, Israel) with the Profilesearch program (Gribskov et al., 1987)identified human EST AI085007, mouse EST AA266872 and eventually,theidentification of a novel hemopoietic cytokine. The cytokine wasinitially referred to as IL-D80, but is also known as p28 according toits apparent molecular mass as determined by SDS-PAGE.

III. Cellular Expression of IL-D8 or IL-27

An appropriate probe or primers specific for cDNA encoding primateIL-D80 or IL-27 are prepared. Typically, the probe is labeled, e.g., byrandom priming.

Southern Analysis: DNA (5 μg) from a primary amplified cDNA library wasdigested with appropriate restriction enzymes to release the inserts,run on a 1% agarose gel and transferred to a nylon membrane (Schleicherand Schuell, Keene, N.H.).

Samples for human mRNA isolation may include: peripheral bloodmononuclear cells (monocytes, T cells, NK cells, granulocytes, B cells),resting (T100); peripheral blood mononuclear cells, activated withanti-CD3 for 2, 6, 12 h pooled (T101); T cell, TH0 clone Mot 72, resting(T102); T cell, THO clone Mot 72, activated with anti-CD28 and anti-CD3for 3, 6, 12 h pooled (T103); T cell, TH0 clone Mot 72, anergic treatedwith specific peptide for 2, 7, 12 h pooled (T104); T cell, TH1 cloneHY06, resting (T107); T cell, TH1 clone HY06, activated with anti-CD28and anti-CD3 for 3, 6, 12 h pooled (T108); T cell, TH1 clone HY06,anergic treated with specific peptide for 2, 6, 12 h pooled (T109); Tcell, TH2 clone HY935, resting (T110); T cell, TH2 clone HY935,activated with anti-CD28 and anti-CD3 for 2, 7, 12 h pooled (T111); Tcell tumor lines Jurkat and Hut78, resting (T117); T cell clones, pooledAD130.2, Tc783.12, Tc783.13, Tc783.58, Tc782.69, resting (T118); T cellrandom γδ T cell clones, resting (T119); CD28-T cell clone; Splenocytes,resting (B100); Splenocytes, activated with anti-CD40 and IL-4 (B101); Bcell EBV lines pooled WT49, RSB, JY, CVIR, 721.221, RM3, HSY, resting(B102); B cell line JY, activated with PMA and ionomycin for 1, 6 hpooled (B103); NK 20 clones pooled, resting (K100); NK 20 clones pooled,activated with PMA and ionomycin for 6 h (K101); NKL clone, derived fromperipheral blood of LGL leukemia patient, IL-2 treated (K106);hematopoietic precursor line TF1, activated with PMA and ionomycin for1, 6 h pooled (C100); U937 premonocytic line, resting (M100); U937premonocytic line, activated with PMA and ionomycin for 1, 6 h pooled(M101); elutriated monocytes, activated with LPS, IFNγ, anti-IL-10 for1, 2, 6, 12, 24 h pooled (M102); elutriated monocytes, activated withLPS, IFNγ, IL-10 for 1, 2, 6, 12, 24 h pooled (M103); elutriatedmonocytes, activated with LPS, IFNγ, anti-IL-10 for 4, 16 h pooled(M106); elutriated monocytes, activated with LPS, IFNγ, IL-10 for 4, 16h pooled (M107); elutriated monocytes, activated LPS for 1 h (M108);elutriated monocytes, activated LPS for 6 h (M109); DC 70% CD1a+, fromCD34+ GM-CSF, TNF_(—)12 days, resting (D101); DC 70% CD1a+, from CD34+GM-CSF, TNF_(—)12 days, activated with PMA and ionomycin for 1 hr(D102); DC 70% CD1a+, from CD34+ GM-CSF, TNF_(—)12 days, activated withPMA and ionomycin for 6 hr (D103); DC 95% CD1a+, from CD34+ GM-CSF,TNF_(—)12 days FACS sorted, activated with PMA and ionomycin for 1, 6 hpooled (D104); DC 95% CD14+, ex CD34+ GM-CSF, TNF_(—)12 days FACSsorted, activated with PMA and ionomycin 1, 6 hr pooled (D105); DC CD1a+CD86+, from CD34+ GM-CSF, TNFα 12 days FACS sorted, activated with PMAand ionomycin for 1, 6 h pooled (D106); DC from monocytes GM-CSF, IL-4 5days, resting (D107); DC from monocytes GM-CSF, IL-4 5 days, resting(D108); DC from monocytes GM-CSF, IL-4 5 days, activated LPS 4, 16 hpooled (D109); DC from monocytes GM-CSF, IL-4 5 days, activated TNFα,monocyte supe for 4, 16 h pooled (D110); epithelial cells, unstimulated;epithelial cells, IL-1β activated; lung fibroblast sarcoma line MRC5,activated with PMA and ionomycin for 1, 6 h pooled (C101); kidneyepithelial carcinoma cell line CHA, activated with PMA and ionomycin for1, 6 h pooled (C102).

A rodent counterpart, e.g., mouse, has been identified, and itsdistributions will be similarly evaluated. Samples for mouse mRNAisolation can include: resting mouse fibroblastic L cell line (C200);Braf:ER (Braf fusion to estrogen receptor) transfected cells, control(C201); Mel14+ naive T cells from spleen, resting (T209); Mel14+ naive Tcells from spleen, stimulated with IFN_, IL-12, and anti IL-4 topolarize to TH1 cells, exposed to IFNγ and IL-4 for 6, 12, 24 h, pooled(T210); Mel14+ naive T cells from spleen, stimulated with IL-4 and antiIFNγ to polarize to Th2 cells, exposed to IL-4 and anti IFNγ for 6, 13,24 h, pooled (T211); T cells, TH1 polarized (Mel14 bright, CD4+ cellsfrom spleen, polarized for 7 days with IFN-_and anti IL-4; T200); Tcells, TH2 polarized (Mel14 bright, CD4+ cells from spleen, polarizedfor 7 days with IL-4 and anti-IFN-_; T201); T cells, highly TH1polarized 3× from transgenic Balb/C (see Openshaw, et al. (1995) J. Exp.Med. 182:1357-1367; activated with anti-CD3 for 2, 6, 24 h pooled;T202); T cells, highly TH2 polarized 3× from transgenic Balb/C(activated with anti-CD3 for 2, 6, 24 h pooled (T203); T cells, highlyTH1 polarized 3× from transgenic C57 bl/6 (activated with anti-CD3 for2, 6, 24 h pooled; T212); T cells, highly TH2 polarized 3× fromtransgenic C57 bl/6 (activated with anti-CD3 for 2, 6, 24 h pooled;T213); T cells, highly TH1 polarized (naive CD4+ T cells from transgenicBalb/C, polarized 3× with IFN_, IL-12, and anti-IL-4; stimulated withIGIF, IL-12, and anti IL-4 for 6, 12, 24 h, pooled); CD44− CD25+ pre Tcells, sorted from thymus (T204); TH1 T cell clone D1.1, resting for 3weeks after last stimulation with antigen (T205); TH1 T cell clone D1.1,10_g/ml ConA stimulated 15 h (T206); TH2 T cell clone CDC35, resting for3 weeks after last stimulation with antigen (T207); TH2 T cell cloneCDC35, 10_g/ml ConA stimulated 15 h (T208); unstimulated B cell lineCH12 (B201); unstimulated mature B cell leukemia cell line A20 (B200);unstimulated large B cells from spleen (B202); B cells from totalspleen, LPS activated (B203); metrizamide enriched dendritic cells fromspleen, resting (D200); dendritic cells from bone marrow, resting(D201); unstimulated bone marrow derived dendritic cells depleted withanti B220, anti CD3, and anti Class II, cultured in GM-CSF and IL-4(D202); bone marrow derived dendritic cells depleted with anti B220,anti CD3, and anti Class II, cultured in GM-CSF and IL-4, stimulatedwith anti CD40 for 1, 5 d, pooled (D203); monocyte cell line RAW 264.7activated with LPS 4 h (M200); bone-marrow macrophages derived with GMand M-CSF (M201); bone-marrow macrophages derived with GM-CSF,stimulated with LPS, IFN_, and IL-10 for 24 h (M205); bone-marrowmacrophages derived with GM-CSF, stimulated with LPS, IFN_, and antiIL-10 for 24 h (M206); peritoneal macrophages (M207); macrophage cellline J774, resting (M202); macrophage cell line J774+LPS+anti-IL-10 at0.5, 1, 3, 6, 12 h pooled (M203); macrophage cell line J774+LPS+IL-10 at0.5, 1, 3, 5, 12 h pooled (M204); unstimulated mast cell lines MC-9 andMCP-12 (M208); immortalized endothelial cell line derived from brainmicrovascular endothelial cells, unstimulated (E200); immortalizedendothelial cell line derived from brain microvascular endothelialcells, stimulated overnight with TNFα (E201); immortalized endothelialcell line derived from brain microvascular endothelial cells, stimulatedovernight with TNFα (E202); immortalized endothelial cell line derivedfrom brain microvascular endothelial cells, stimulated overnight withTNFα and IL-10 (E203); total aorta from wt C57 bl/6 mouse; total aortafrom 5 month ApoE KO mouse (X207); total aorta from 12 month ApoE KOmouse (X207); wt thymus (O214); total thymus, rag-1 (O208); totalkidney, rag-1 (O209); total kidney, NZ B/W mouse; and total heart, rag-1(O202). High signal was detected in the monocyte cell line RAW 264.7activated with LPS 4 h (M200); T cells, highly TH1 polarized 3× fromtransgenic C57 bl/6 (activated with anti-CD3 for 2, 6, 24 h pooled;T212); and T cells, highly TH1 polarized (naive CD4+ T cells fromtransgenic Balb/C, polarized 3× with IFNγ, IL-12, and anti-IL-4;stimulated with IGIF, IL-12, and anti IL-4 for 6, 12, 24 h, pooled).

IV. Chromosome Mapping of IL-D80

An isolated cDNA encoding the IL-D80 is used. Chromosome mapping is astandard technique. See, e.g., BIOS Laboratories (New Haven, Conn.) andmethods for using a mouse somatic cell hybrid panel with PCR. The humanIL-D80 gene is located on chromosome 16p11.

V. Expression and Purification of IL-D80 or IL-27 Proteins

Multiple transfected cell lines are screened for one which expresses thecytokine at a high level compared with other cells. Various cell linesare screened and selected for their favorable properties in handling.Natural IL-D80 can be isolated from natural sources, or by expressionfrom a transformed cell using an appropriate expression vector.Purification of the expressed protein is achieved by standardprocedures, or may be combined with engineered means for effectivepurification at high efficiency from cell lysates or supernatants. FLAGor His₆ segments can be used for such purification features.Alternatively, affinity chromatography may be used with specificantibodies, see below.

cDNAs encoding full length human and mouse IL-D80 were cloned into thepCDM8-E-tag vector via HindIII-XhoI (h/mp28-E). EBI3: human and mouseEBI3 were cloned into pME18S-Ig vector via EcoRI/XhoI (h/mEBI3-Ig) andthe mature portion of human EBI3 into p-FLAG-CMV-1 vector viaHindIII-NotI (F-hEBI3). One chain fusions EBI3/p28: HindIII-XbaIfragments were generated encoding the mature part of human or mouseEBI3, followed by the synthetic linker GSGSGGSGGSGSGKL (SEQ ID NO:13)and by the mature coding sequence of human or mouse IL-D80 viaHindIII-NotI. Fragments were inserted into pFLAG-CMV-1 (Sigma, St.Louis, Mo.) using HindIII-NotI sites.

WSX-1/TCCR: the preprotrypsin leader peptide and the FLAG-tag encodingpart of p-FLAG-CMV-1 vector were deleted by PCR, instead an RGSH₆-tagwas introduced via SalI/SmaI (pCMV-1-RGSH₆); the cDNA encoding theextracellular part of human WSX-1 was cloned into this vector viaHindIII-SalI (soluble hWSX-1-R). In general restriction sites wereintroduced through the respectively used PCR primers and cDNA wasamplified using standard PCR protocols. Proteins were produced viatransient expression in HEK293T cells. For experiments requiring pureproteins purification was performed by affinity chromatography using therespective protein tags.

VI. Transient Transfection, Metabolic Labeling and Immunoprecipitation

1×10⁶ HEK293T cells were transiently transfected with a total amount of5 μg plasmid DNA (control vector, expression vectors encoding h/m p28-E,F-hEBI3 and mEBI3-Ig, or respective combinations). Cells were culturedfor 24 hr after transfection, then metabolically labeled for 16 hr with50 μCi/ml Pro-mix L-[³⁵S] in vitro cell labeling mix (AmershamPharmacia, Piscataway, N.J.) in cysteine/methionine free MEM. Proteinswere precipitated from supernatants with either anti-FLAG M2 agarose(Sigma, St. Louis, Mo.), with anti-E-tag mAb bound to protein GSepharose® (Amersham Pharmacia), or with protein A Sepharose® (AmershamPharmacia).

VII. Retroviral Constructs

The mature part of human and mouse WSX-1 was cloned into pMX vector viaHindIII-NotI, then a sequence encoding the preprotrypsin leader peptidefused to a FLAG epitope was cloned into the vector in frame and 5′ ofWSX-1 via BamHI-HindIII (F-h/mWSX-1). Retrovirus obtained bytransfection of BOSC23 cells was used to infect parental Ba/F3 cells andcell surface expression of the desired proteins was monitored using aFLAG-PE-staining in FACS analysis.

VIII. Isolation of Homologous IL-D80 Genes

The IL-D80 cDNA, or other species counterpart sequence, can be used as ahybridization probe to screen a library from a desired source, e.g., aprimate. cell cDNA library. Many different species can be screened bothfor stringency necessary for easy hybridization, and for presence usinga probe. Appropriate hybridization conditions will be used to select forclones exhibiting specificity of cross hybridization.

Screening by hybridization using degenerate probes based upon thepeptide sequences will also allow isolation of appropriate clones.Alternatively, use of appropriate primers for PCR screening will yieldenrichment of appropriate nucleic acid clones.

Similar methods are applicable to isolate either species, polymorphic,or allelic variants. Species variants are isolated using cross-specieshybridization techniques based upon isolation of a full length isolateor fragment from one species as a probe.

Alternatively, antibodies raised against human IL-D80 or IL-27 will beused to screen for cells which express cross-reactive proteins from anappropriate, e.g., cDNA library. The purified protein or definedpeptides are useful for generating antibodies by standard methods, asdescribed above. Synthetic peptides or purified protein are presented toan immune system to generate monoclonal or polyclonal antibodies. See,e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene; andHarlow and Lane (1989) Antibodies: A Laboratory Manual Cold SpringHarbor Press. The resulting antibodies are used for screening,purification, or diagnosis, as described.

IX. Preparation of antibodies specific for IL-D80 or IL-27

Synthetic peptides or purified protein are presented to an immune systemto generate monoclonal or polyclonal antibodies. See, e.g., Coligan(1991) Current Protocols in Immunology Wiley/Greene; and Harlow and Lane(1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press.Polyclonal serum, or hybridomas may be prepared. In appropriatesituations, the binding reagent is either labeled as described above,e.g., fluorescence or otherwise, or immobilized to a substrate forpanning methods. Immunoselection, absorptions, and related techniquesare available to prepare selective reagents, e.g., exhibiting thedesired spectrum of selectivity for binding.

X. Generation and Analysis of Genetically Altered Animals

Transgenic mice can be generated by standard methods. Such animals areuseful to determine the effects of deletion of the gene, in specifictissues, or completely throughout the organism. Such may provideinteresting insight into development of the animal or particular tissuesin various stages. Moreover, the effect on various responses tobiological stress can be evaluated. See, e.g., Hogan, et al. (1995)Manipulating the Mouse Embryo: A Laboratory Manual (2d ed.) Cold SpringHarbor Laboratory Press.

IX. Expression/Distribution of IL-27

cDNAs from various libraries or cultured macrophages and dendritic cellswere prepared as described (see, e.g., Bolin, et al. (1997) J. Neurosci.17:5493-5502) and used as templates for quantitative PCR. 50 ng cDNA wasanalyzed for expression of human and mouse p28 and EBI3 by thefluorogenic 5′-nuclease PCR assay (see, e.g., Holland, et al. (1991)Proc. Natl. Acad. Sci. 88:7276-7280) using the ABI Prism 7700 SequenceDetection System (Perkin-Elmer, Foster City, Calif.). Analysis of cDNAsamples was corrected for expression of 18S rRNA using a VIC labeledprobe (Perkin-Elmer) in multiplex reactions.

Analysis of a large panel of human and mouse cDNA libraries by real timequantitative PCR showed that expression of IL-D80 and EBI3 is highlyrestricted. Both mRNAs are primarily found in cells of myeloid lineagein human as well as mouse. Highest levels of human mRNA's were found inLPS activated monocytes and monocyte derived dendritic cells (DCs). Avery high level of hEBI3 MRNA but not hp28, was seen in placenta. Thisobservation is in agreement with earlier reports of high levels of EBI3protein in placental syncytiotrophoblasts [Devergne, 1997 #3]. A similarpattern emerged when we analyzed the expression profile of mouse IL-D80and EBI3. Although mEBI3 was also expressed in some T and B celllibraries, highest levels of both mIL-D80 and mEBI3 was in activatedmacrophages.

Since antigen presenting cells are also the primary source of IL-12(see, e.g., Macatonia, et al. (1995) J. Immunol. 154:5071-5079) westudied the kinetics of production of IL-12p35, IL-12p40, IL-D80 andEBI3 by monocyte derived DCs stimulated with LPS. Human monocytes wereisolated from peripheral blood, stimulated with GM-CSF and IL-4 for 7days to obtain immature DCs. Subsequently, these CD14+CD11c+DCs wereactivated by LPS for various time intervals and mRNA levels of IL-12p35,IL-12p40, IL-D80 and EBI3 were analyzed by real time quantitative PCR.Despite substantial variations in the absolute amounts of PCR productfrom donor to donor and from protein to protein, the kinetics recordedwere consistent and revealed subtle differences between the fourinvestigated proteins. After an initial lag phase, message levels forIL-12p35 and IL-12p40 rapidly increased and consistently peaked between8 and 14 hours of LPS stimulation, then dropped back to background levelafter 24 hours. The profiles for the two subunits of IL-12 areessentially superimposable. A very transient expression was alsoobserved for IL-D80, although maximal message levels were already foundafter 3-6 hours. Similar to IL-12, mRNA levels for p28 declined tobackground levels after 24 hours. In contrast, EBI3 showed lesstransient expression although its transcription was also rapidly inducedas early as 3 h after LPS stimulus. Reaching maximal EBI3 mRNA levelsbetween 12 and 24 hours, after 72 hours EBI3 message in all three donorswas still above the unstimulated background levels.

X. Transient Transfection, Metabolic Labeling, and Immunoprecipitation

Appropriate host cells were transiently transfected with empty vectorsor expression vectors encoding hIL-D80E (E=E-tagged) and/or FhEBI3(F=FLAG-tagged). Cells were cultured to 24 hrs. and then metabolicallylabeled for 16 hrs with 50 μCi/ml PRO-MIX L-[³⁵S] in vitro cell labelingmix (Amersham Pharmacia) in cysteine/methionine free MEM cell culturemedia. Proteins were precipitated from 300 mL supernatant with eitherthe anti-His5 mAb or anti-E or anti-F mAb. The IL-12R like subunit,WSX-1/TCCR, was also detectably labeled with RGSH₆-tag (shNR30R) andimmunoprecipated as above.

XI. 2D-PAGE

Purified labeled IL-27 composite cytokine or IL-27-WSX-1/TCCR complexwere run on a nonreducing 10% NUPAGE gel in MES running buffer (Novex,San Diego, Calif.). Appropriate lanes were excised, reduced in samplebuffer containing DTT, laid horizontally on two-well 10% gels, and runreduced in a second dimension. One gel was silver stained (Daiichi,Tokyo, Japan) while the other was blotted to a PVDF membrane anddeveloped using appropriate mAbs. It was found that hIL-80E could beco-immunoprecipitated with shNR30R in the presence of FhEBI3 using theanti-His₆ mAb. Alternatively, shNR30R could be immunoprecipated in thepresence of hIL-80E and FhEBI3 using the anti-E mAb or anti-F mAb.

XII. Biological Effects of IL-27

A. Naive Human and Mouse T cells

CD4+CD45RB^(high) or CD4+CD45RB^(low) T cell subsets were purified fromthe spleen and mesenteric lymph nodes of >6 month old IL-10-/-C57/B6 N12mice as described (Davidson et al. (1998) J. Immunol. 161:3143-3149).Cells were fractionated into CD4+CD45RB^(high) and CD4+CD45RB^(low) cellpopulations by two color sorting on a FACSTAR plus (Becton Dickinson,San Jose, Calif.). All populations were >99% pure upon reanalysis.CD4+CD45RB^(high) or CD4+CD45RB^(low) were put into a proliferationassay with plate bound anti-CD3 (145.2C11) stimulation as described(Davidson et al. (1998) J. Immunol. 161:3143-3149). Additions to thegrowth media included anti-IL-2 Mab (JES6-1A12) 100 μg/ml, and cytokinesas indicated. Cells were incubated for 5 days in a humidified chamber(37° C., 5% CO₂) with [³H]TdR (Amersham) added at a final concentrationof 1 μCi/well for the last 24 h of incubation.

Sorted mouse naive T cells (CD4+CD45RB^(high)) and memory/activated Tcells (CD4+CD45RB^(low)) were stimulated with CD3 mAb for four days inthe presence of anti-IL-12 antibody and various amounts of mIL-27. Uponstimulation, naive T cells, but not memory T cells, showed a strongproliferative response. Proliferation was augmented by addition of IL-12at saturating levels, revealing synergy between IL-27 and IL-12 onunstimulated T cells. IL-27 was able to act as a strong expansion factorfor anti-CD3, anti-CD28 activated naive T cells in the absence of IL-12.

FACS purified CD45RA and CD45RO T cells (purity >99%) were cultured at adensity of 4×10⁴ cells/well in a 96-well plate previously coated withanti-CD3 antibody at 10 μg/ml and soluble anti-CD28 at 1 μg/ml with orwithout IL-26/EBI3. Anti-hIL-2 mAb 17H12 and anti-hIL-2R mAb B-B10(Diaclone, Besabcibm France) were added at 10 μg/ml where indicated.IL-27 was also able to induce proliferation of FACS sorted human CD45RAnaive T cells isolated from peripheral blood mononuclear cells (PBMC).Similar to the results with mouse naive T cells, IL-27 induced strongproliferation of CD3/CD28 naive T cells in the presence of anti-IL-2.This response was enhanced by the addition of IL-12. No response wasseen with IL-27 treated CD45RO memory cells.

Thus, IL-27 dependent. proliferation can be enhanced by costimulatorysignals through either CD28 or the IL-12 receptors. IL-27 inducedproliferation is dependent on simultaneous crosslinking of CD3/TCR,since no proliferation was observed in the absence of CD3 activation(data not shown). The same maximal proliferative response could beinduced by stimulation with conditioned medium of p28/EBI3co-transfected cells (data not shown). To compare the abilities of IL-27and IL-12 to induce proliferation of naïve CD4+ T cells, FACS sortedmouse CD4+CD45Rbhigh T cells were pre-cultured with plate bound anti-CD3mAb, and either IL-27 or IL-12 were titrated into the cultures. IL-27proved to be a much more potent proliferative stimulus for these cells(FIG. 4C).

Thus, IL-27 dependent proliferation can be enhanced by costimulatorysignals through either CD28 or the IL-12 receptors. IL-27 inducedproliferation is dependent on simultaneous crosslinking of CD3/TCR,since no proliferation was observed in the absence of CD3 activation.The same maximal proliferative response could be induced by stimulationwith conditioned medium of IL-27 co-transfected cells. To compare theabilities of IL-27 and IL-12 to induce proliferation of naïve CD4+ Tcells, FACS sorted mouse CD4+CD45RB^(high) T cells were pre-culturedwith plate bound anti-CD3 mAb, and either IL-27 or IL-12 were titratedinto the cultures. IL-27 proved to be a much more potent proliferativestimulus for these cells.

B. Induction of IFN-γ

The ability of human and mouse IL-27 to induce the production of IFNγ_inthe presence of a neutralizing anti-IL-2 mAb, with costimulation viaanti-CD3 or anti-CD3/anti-CD28 and both in the absence and presence ofIL-12 was measured. In this assay neither hIL-27 nor hIL-12 by itselfinduced IFNγ_production in anti-CD3 or anti-CD3/anti-CD28 activatedCD4⁺CD45RA T cells. IFNγ production was only observed in the presence ofboth cytokines indicating strong synergy between IL-27 and IL-12.

Sorted mouse CD4⁺CD45RB^(high) naïve T cells were stimulated for 4 dayswith anti-CD3 mAb alone or with anti-CD3 mAb/anti-CD28 mAb andsaturating amounts of IL-27 and IL-12. In the absence of anti-CD28costimulation neither IL-27 nor IL-12 by itself was capable of inducingsubstantial amounts of IFNγ_. However, the combination of IL-27 andIL-12 induced up to about 300 ng/ml of IFNγ_. With anti-CD3/anti-CD28costimulation, IL-27 as well as IL-12 were capable of inducing IFNγproduction. The combination of both factors led to an additive effectwith IFNγ levels up to 550 ng/ml.

C. IL-27 does not drive Th2 polarization of naïve T cells

Sorted mouse CD4⁺CD45RBhigh T cells were cultured with plate boundanti-CD3 and anti-CD28 in the presence of IL-4 and IL-27. IncludingIL-27 in the cultures led to a decreased IL-13 production both in theabsence and presence of IL-4. Thus, while inducing a strong Th1response, IL-27 does not appear to promote Th2 polarization.

D. IL-27 binds to WSX-1/TCCR

Because of the relationship between IL-27 and the IL-6/IL-12 family, thesearch for the signaling receptors was concentrated on this family.Members of this family were introduced into BaF3 cells and tested forbinding to IL-27. Of the receptors tested only Ba/F3 cells expressingthe orphan cytokine receptor WSX-1/TCCR (see, e.g., Sprecher, et al.(1998) Biochem. Biophys. Res. Comm. . 246:82-90; and Chen, et al. (2000)Nature 407:916-920) showed binding to tagged IL-27. BaF3 cells infectedwith retroviral constructs expressing either F-tagged human or mouseWSX-1 cDNA (F-hWSX-1 or F-mWSX-1) showed cellular staining usinganti-FLAG mAb. Cells expressing F-hWSX-1 were then incubated with eitherhEBI3-Ig alone or with coexpressed hIL-D80-E and EBI3-Ig for two hours.Heterodimeric IL-D80/EBI3 bound to WSX-1 while EBI3-Ig itself showed nodetectable binding. Similarly, only the combination of mIL-D80-E andmEBI3-Ig provided a detectable interaction with mWSX-1-expressing BaF3cells, whereas the two individual proteins were not able to do so.Incubation of independently expressed mIL-D80-E and mEBI3-Ig withF-mWSX-1 expressing BaF3 cells also led to cellular staining.Untransfected control cells were not stained by IL-D80/EBI3,demonstrating the specificity of the observed interactions.

These results were confirmed by co-immunoprecipitation experiments usinga soluble extracellular form of hWSX-1 with a C-terminal RSGH₆-tag (R).Proteins from supernatants of transiently transfected HEK293T cellscontaining F-hEBI3 or coexpressed hILD80-E/F-hEBI3 wereimmunoprecipitated using either FLAG M2-agarose, protein GSepharose®-coupled anti-E-tag mAb(Amersham Pharmacia, Piscataway, N.J.)or protein G Sepharose®-coupled anti-H₅ mAb. The primary precipitateswere washed and then incubated with HEK293T cell supernatants containingshWSX-1-R. Secondary precipitates were separated by SDS-PAGE andsubjected to western blot. Precipitated proteins were visualized byenhanced chemiluminescence (ECL) 5 using antibodies against therespective protein tags. Only when all three proteins were present(hlL-D80-E, F-hEBI3 and shWSX-1-R), immunoprecipitation of one proteinbrought down both other components independently of theimmunoprecipitating antibody used. The same co-immunoprecipitationexperiment using the respective mouse orthologues had similar results.

To address the question if WSX-1 was sufficient to mediate IL-27 signaltransduction, proliferation of BaF3 cells expressing human or mouseWSX-1 was tested. These cells proliferate in response to IL-3 but didnot proliferate in response to IL-27. Thus WSX-1 appears to be requiredbut not sufficient for IL-27 mediated signal transduction. Theidentification of additional IL-27 signal transducing receptor subunitsis currently in progress.

All references cited herein are incorporated herein by reference to thesame extent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety for all purposes.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

1. An isolated polypeptide comprising the amino acid sequence of SEQ IDNO:2.
 2. The isolated polypeptide of claim 1, wherein the polypeptide ischemically modified.
 3. The isolated polypeptide of claim 2, wherein thechemical modification is glycosylation or phosphorylation.